"The Islamic Republic of Iran, based on its principled policy of condemning terrorism and its strong will to seriously confront this evil phenomenon, condemns the recent terrorist attack in Orlando," foreign ministry spokesman Hossein Jaberi Ansari said, quoted by state news agency IRNA.
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In Iran, whose diplomatic ties with the United States have been severed for more than three decades, homosexuality is banned and can be punishable by death, under the Islamic Sharia laws in force since its 1979 revolution.
Forty-nine people were killed and 53 others wounded when a heavily armed gunman opened fire and seized hostages on Sunday at the Pulse gay nightclub, in the deadliest mass shooting in US history.
The assailant, Omar Mateen, 29, a US citizen of Afghan origin, reportedly made a 911 call during the massacre pledging allegiance to IS, which itself claimed responsibility for the attack, calling Mateen "one of the soldiers of the caliphate".