The US says it has intelligence that Hamas has a command centre under Al-Shifa hospital in Gaza City.
National Security spokesman John Kirby said the group stored arms there and were prepared for an Israeli attack.
This is the first time the US has independently backed claims by close ally Israel that Hamas uses hospitals to hide its bases. Hamas denies this.
The statement came as Israel faced mounting global pressure to protect civilians trapped in the hospitals.
US President Joe Biden said that Al-Shifa hospital "must be protected" from intense fighting around the complex, and UK Prime Minister Rishi Sunak said Israel must act within international law.
The area around Al-Shifa, Gaza's biggest hospital, has become the focus of fighting in recent days. Thousands of people are believed to be sheltering there.
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Mr Kirby said the US had its own intelligence, which had come from a variety of sources, suggesting that Hamas and Islamic Jihad used hospitals in the Gaza Strip and tunnels underneath them to conceal military operations and hold hostages.
Previously the US administration had only cited open-source intelligence and would not confirm that it had its own sources for this.
"Hamas and Palestinian Islamic Jihad members operate a command and control node from Al-Shifa in Gaza City," he said.
"They have stored weapons there and they are prepared to respond to an Israeli military operation against that facility."
Mr Kirby said this showed how challenging the Israeli operation was as "Hamas has deeply embedded itself within the civilian population".
"To be clear, we do not support striking a hospital from the air and we do not want to see a firefight in the hospital where innocent people, helpless people, sick people are simply trying to get the medical care that they deserve, not to be caught in a crossfire. Hospitals and patients must be protected," he said.
"As we have been clear on multiple occasions, Hamas's actions do not lessen Israel's responsibility to protect civilians in Gaza, and this is obviously something we're going to have an active conversation with our counterparts about."
Responding to the White House's remarks on Tuesday, Hamas said in a statement reported by the AFP news agency that Israel would use Washington's assessment to give it a "green light" to commit "brutal massacres" against medical facilities, "destroying Gaza's healthcare system and displacing Palestinians".
Doctors inside Al-Shifa say that dozens of people, including at least three premature babies, have died due to a lack of fuel, medicine and power.
They have told the BBC that more than 100 bodies lay unburied in courtyards, and anyone trying to leave the complex - or even move between buildings - risks death due to the violence in the area.
Israel maintains that it has not fired directly on hospitals in Gaza despite what it says is Hamas's presence in them. The Israeli military has vowed to destroy Hamas, a designated terror group in the US, UK and EU, in the Gaza Strip.
On Monday Israel released video of what it said was a Hamas hideout under the Rantisi children's hospital in northern Gaza City.
In a six-minute video marked as "raw footage", the IDF's chief spokesman Rear Admiral Daniel Hagari is seen saying he is at a tunnel 200 metres away from Rantisi.
In the video, Hagari points to the tunnel entrance, which he says he believes is "connected" to the hospital, and electrical wiring he says proved solar panels had been powering a bunker.
No further evidence is provided for the existence of a command centre but the IDF spokesman said investigations were continuing.
The footage then cuts to basement rooms he says are inside Rantisi hospital. He points to a cache of weapons, including explosive belts and grenades, and a motorcycle with what he says is bullet damage.
Rear Adm Hagari says there are signs that hostages kidnapped by Hamas during the group's attack on Israel had been held in the basement.
A shot shows a chair with what appears to be rope ties, below a control panel marked "World Health Organization" with a child's feeding bottle resting on top.
In another room, he points to a calendar on the wall counting the days in Arabic from 7 October - the date of Hamas's attack into southern Israel.
Rear Adm Hagari claims the calendar shows the "terrorists' shifts" as they guarded the room. He says names were written alongside shift times.
The top of this document mentions the "al-Aqsa flood" - Hamas's codename for the 7 October attacks. However, the Arabic words translate to the days of the week, not names.
He separately states that the room is decorated with curtains like a video studio. Of the hostage videos released by Hamas and Palestinian Islamic Jihad, none have shown a matching pattern in the background.
The IDF escorted CNN journalists around the site and the hospital, which was evacuated on Friday. BBC News has not visited the site and is not able to independently verify any of the allegations made by Hagari.
However, the Hamas-run Health Ministry in Gaza responded in detail to the Israeli claims, describing them as a "theatrical farce".
It said the underground space shown in the video was used for administration and storage, and as a shelter from air strikes.
It described the display of weapons as staged, and said there were thousands of similar motorcycles in Gaza.
The ministry says that more than 11,000 people have been killed in Israel's operations against Hamas, since the group killed 1,200 people in Israel on 7 October and took about 240 people hostage.
Additional reporting by BBC Verify's Paul Brown.