Iran responded with attacks targeting several countries in the Gulf, including Bahrain, Kuwait, Qatar and Oman.
The US military's Central Command said it hit some 140 targets in the Sunday morning strikes, far more than the last two rounds, and went after missile and drone launch sites, ammunition dumps, communication equipment and other sites.
The attacks "degrade Iran's ability to attack civilian mariners and commercial vessels freely transiting the strait", it said.
The new crossfire in the Persian Gulf comes days after US President Donald Trump suggested an interim deal in the Iran war was "over".
US Defence Secretary Pete Hegseth wrote online: "Iran made a poor choice. Now they pay."
Mohammad Bagher Qalibaf, the speaker of Iran's parliament and a main negotiator, responded.
"The era of one-sided deals is OVER," he wrote on X. "We told you: keep your word or pay the price. Reality is knocking."
The US launched three rounds of airstrikes targeting Iran in the last week over Iranian attacks on ships heading through the Strait of Hormuz using a route seeking to avoid the Islamic Republic's territorial waters.
Tehran retaliated by attacking nations in the region hosting US military forces, while insisting it alone must control the strait and potentially charge vessels for travelling through it.
Iran's paramilitary Revolutionary Guard said they had destroyed a command and control centre and drone hangars at US ally Jordan, targeted a US radar site in Kuwait, attacked American aircraft carrier support and refuelling platforms in Oman and destroyed a jet maintenance centre and command facility in Qatar.
Missile alerts sounded across several Gulf Arab nations.
Qatar's military said in a statement it intercepted incoming Iranian fire, with explosions heard in neighbouring United Arab Emirates.
Three people, including a child, were wounded as a result of falling shrapnel from the interception of Iranian attacks, Qatar's Interior Ministry said.
Meanwhile, missile alerts sounded for the third time on Sunday in Bahrain, an island kingdom in the Persian Gulf home to the US Navy's 5th Fleet. Kuwait's military also said it was intercepting incoming fire.
The Omani state news agency said drones struck sites in northeastern Oman, in the area that sits on the Strait of Hormuz. The attack came after Oman and Iran held talks on Saturday.
The UAE said the attacks on Sunday were outside the country's borders, although sirens sounded in the morning.
In the Strait of Hormuz attack, a Cyprus-flagged container ship was hit by Iran and suffered "significant engineroom damage" and a civilian crew member is missing, US Central Command said early on Sunday morning.
The Indian Ministry of External Affairs said an Indian national was missing after an attack on the commercial vessel GFS Galaxy.
The United Kingdom Maritime Trade Operations centre, overseen by the British military, said the ship had been travelling on a route hugging the shoreline of Oman.
That's been the way ships have entered and exited the Persian Gulf while avoiding Iranian territorial waters. The ship's crew abandoned the vessel as it was ablaze, the centre said.
Iran said that the strait would remain closed until further notice and said it would consider targeting "additional enemy bases in the region" if it faced more attacks.
Iran's new supreme leader, still unseen since the war began, also vowed in his first statement since the funeral of his father, Ayatollah Ali Khamenei, that Iranians would avenge his killing in the war's opening strikes on February 28.
Such revenge "is the will of our nation and must certainly be carried out", Supreme Leader Ayatollah Mojtaba Khamenei said in a statement carried on state television.
with Reuters
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