The new US assault across multiple cities comes as efforts to negotiate an end the war again appeared stuck, with Iran insisting it would maintain its chokehold on the Strait of Hormuz.
Explosions from the strikes echoed around Iran's capital, Tehran, as well as the port city of Bandar Abbas and other southern areas along the Strait of Hormuz.
After the first round of American strikes, Iran launched missiles at Bahrain, Kuwait and Jordan.
Kuwait closed its airspace as its air defences fought off the Iranian attack.
Kuwait's Directorate General of Civil Aviation said flights were being diverted to other airports, without elaborating.
"This measure comes in light of the state of Kuwait being subjected to sinful Iranian aggressions and the potential risks that may result from this on civil aviation traffic in the region," Kuwait said.
Iran's United Nations envoy said the US should refrain from threats of force if it wants a deal.
"Iran has never negotiated under threats and pressure and will never submit to pressure or question," Ambassador Amir Saeid Iravani told the UN Security Council.
Earlier, US Central Command said its latest attacks targeted "military surveillance capabilities, communication systems, and air defence sites across Iran" in response to what it called Tehran's "unwarranted and continued aggression."
"US Marine Corps, Air Force, and Navy assets fired precision munitions on Iranian targets that posed a threat to US forces and international commercial ships transiting regional waters," the command said.
President Donald Trump told Fox News the US strikes would stop shortly but that he would resume heavy bombing if Iran's leaders did not sign an agreement with the US immediately.
Iran's Islamic Revolutionary Guard Corps said they had launched counter-attacks on 18 US military targets at air bases in Kuwait and Bahrain, and Bahrain's interior ministry said sirens were sounded.
The IRGC later said it had also targeted the al-Azraq air base in Jordan for a second night running, firing 12 ballistic missiles at the US base.
Iran's top joint military command warned it would fire on any vessel attempting to pass through the the Strait of Hormuz, which has been largely closed for months. Iranian media reported two ships were fired upon.
Central Command denied the strait was closed, saying commercial ships were still transiting the strait despite Iran's threats.
The US attacks are the latest development in an escalating exchange of strikes that threaten to reignite a full-scale war, which was paused in early April when the two sides agreed to a fragile ceasefire.
US Defence Secretary Pete Hegseth presented the move as an effort to force Iran into a deal to end the conflict.
He said Iran had been given a chance to make a deal but had not taken it.
"We will strike them hard tonight, and hopefully Iran makes a good decision," Hegseth said.
He accused Iran of "choosing to play games" and added: "If we need to negotiate with bombs, we'll negotiate with bombs, and we're very good at it."
The strikes come a day after the US hit Iran following the crash of an Army helicopter near the Strait of Hormuz that Trump blamed on the Islamic Republic.
Trump earlier revealed the United States has been taking oil out of Iran.
"I'm just announcing today for the first time, but we've been taking out millions of barrels of oil, millions of barrels every night," Trump said, adding that Iran "just figured it out".
"Millions of barrels of oil has come out, and that's why it's at $US85-90 a barrel, instead of $US250," Trump said, sharing no other details about these operations.
He later said more than 100 million barrels of crude had passed through the Straits of Hormuz as part of what he called a secret US mission to support oil tankers.
Trump said the United States is still looking to make a deal.
"We want a deal that is meaningful, we want a deal that works," he added.
with AP and DPA
10°C