Tensions between Iran and the West have resurged since President Donald Trump pulled the United States out of world powers' 2015 nuclear deal with Tehran, calling it deeply flawed.
European signatories are scrambling to save the accord, which they see as crucial to forestalling an Iranian nuclear weapon, by seeking to ring-fence trade with Iran against new US financial sanctions to dissuade Tehran from quitting the deal.
In case European, Russian and Chinese signatories to the deal prove unable to protect its economic benefits for Iran, Khamenei said he has ordered Iran's atomic energy agency to be prepared to upgrade its uranium enrichment capacity.
He appeared to be hardening a threat to restore Iran's disputed enrichment drive first made after Trump quit the nuclear deal.
One of Trump's demands - which European allies support in principle - is new negotiations to rein in Iran's ballistic missile program, which was not covered by the nuclear deal. Khamenei again ruled this out.
Under the deal, the Islamic Republic curbed its capacity to enrich uranium for nuclear fuel and in return won a lifting of most international sanctions that had hobbled its economy.
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Trump also objected that the 2015 deal, reached under his predecessor Barack Obama, did not address Iran's nuclear work beyond 2025 or its role in conflicts in Yemen and Syria. Though committed to the deal, European powers share Trump's concerns and want broader talks with Iran to address the issues.
Khamenei rejected this as plans "to use economic, psychological and operational pressures".
Iran's increasing capacity to enrich uranium to higher levels of fissile purity was one of the burning issues during years of negotiations with world powers, which wanted enrichment limited to research scale to minimize the risk of Iran being able to assemble a nuclear explosive at short notice. Iran has repeatedly denied seeking a nuclear weapon from enrichment.
After the US withdrawal on May 12, Iran said it would remain in the nuclear deal only if were protected from renewed US sanctions on key sectors of its economy such as oil, in cooperation with other signatories of the pact.
Options being considered by the EU to keep Tehran in the nuclear deal include new credit lines for Tehran, increased energy cooperation and implementing EU laws to block European companies from caving in to US sanctions.
In his speech marking the 29th anniversary of the death of Iranian revolutionary leader Ayatollah Ruhollah Khomeini, Khamenei insisted on resisting US pressure and again warned of a harsh response if Iran came under attack.
"The enemy knows that if they hit us once they will be hit 10 times more... The Islamic Republic will continue its support for oppressed nations," Khamenei said.
Shi'ite Muslim Iran backs President Bashar al-Assad in Syria's civil war, Shi'ite militias in Iraq, Houthi rebels in Yemen's conflict and Lebanon's heavily armed Hezbollah movement.