“This nation will never surrender,” Khamenei said in a speech read on state television, in which he called Trump’s ultimatum “unacceptable”.
“America should know that any military intervention will undoubtedly result in irreparable damage,” he said.
Khamenei, in power since 1989 and the final arbiter of all matters of state in Iran, had earlier vowed the country would show “no mercy” towards Israel’s leaders.
The speech followed a night of strikes, with Israeli attacks destroying two buildings making centrifuge components for Iran’s nuclear programme near Tehran, according to the UN nuclear watchdog.
“More than 50 Israeli Air Force fighter jets… carried out a series of air strikes in the Tehran area over the past few hours,” the Israeli military said, adding that several weapons manufacturing facilities were hit.
