Rome rejected accusations that the arrests were politically motivated while in Tehran, foreign ministry spokesman Ramin Mehmanparast called the detentions "an indicator of a new game with ambiguous intentions which aimed to create distractions".
On Wednesday, Italy arrested seven people on suspicion of trafficking arms to Iran, including two Iranians they believe are secret agents and five Italians.
One of those held was an Iranian journalist accredited with Rome's foreign press club and two other Iranians, also believed to be spies, are still at large.
Foreign Minister Franco Frattini issued a statement after Italy's ambassador in Iran, Alberto Bradanini, was summoned to Tehran's foreign ministry to hear a protest over the arrests in Italy earlier this week.
"I firmly reject any Iranian insinuation that the recent arrests by the Italian magistrates were politically motivated," Frattini said.
"Italy is founded on principles of law and the magistrature is independent," he said.
Italian media quoted Iranian state television as denouncing the arrests as part of an "American-Zionist plot to unjustly accuse the Islamic Republic".
Italy is one of Iran's main trading partners in Europe but Prime Minister Silvio Berlusconi's close ties with Israel and diplomatic pressure over the nuclear dispute with Tehran have led to a sharp reduction in Italian investments in Iran.
When Berlusconi visited Israeli last month Italian officials announced they wanted to block new oil and gas investments in Iran, where Italy's ENI is involved in the Darkhovin oil field.
Last month Frattini announced a suspension of export credit guarantees for firms investing in Iran.
Iran is subject to an international arms embargo and Berlusconi has said on several occasions that trade sanctions ought to be toughened.
Italian magistrates said the five Italians and four Iranians implicated in the operation were sending weapons to Iran from Italy and via third countries.
(Additional reporting by Ramin Mostafavi in Tehran; Editing by Michael Roddy)
