The American-Israel Public Affairs Committee (AIPAC) said on Wednesday that the FBI visited its offices to request files related to two employees and served four others with subpoenas.
The influential lobbying group said it was cooperating with the investigation.
"We believe any court of law or grand jury will conclude that AIPAC employees have always acted legally, properly and appropriately," it said.
The FBI, which was not immediately available to comment, has been investigating whether a Pentagon analyst passed secret papers to Israel about Iran.
US government sources say the analyst in question is in Defence Secretary Donald Rumsfeld's office and passed the papers through AIPAC.
The FBI first raided AIPAC's offices in August, seizing computer files related to two employees.
Israeli officials have denied spying on the United States.