Committee Chairman Darrell Issa of California and Rep. Jim Jordan of Ohio requested the probe Monday in a letter to Treasury Department Inspector General Russell George.
"We are troubled by evidence that the IRS may have conducted unnecessary audits and systematic post hoc reviews of entire groups of applicants as well as certain groups that have long possessed tax-exempt status," the lawmakers said.
George released a report in May that said the IRS had flagged groups for extra scrutiny based on political ideology, but those findings focused entirely on tax-exemption applicants rather than groups that were already green-lighted.
The audit triggered a political firestorm involving statements of outrage from Democratic and Republican lawmakers, agency apologies, congressional hearings, a Justice Department probe and a personnel shake-up at the IRS.
In recent weeks, however, the IRS issue has sharpened into a partisan fight as Democrats have questioned the veracity of George's report and produced documents they say show that liberal organizations also were targeted but appeared to have been left out of the May audit.
Where Republicans have seen scandal and tried unsuccessfully to directly link the issue to the White House, Democrats have seen an agency simply trying to do its job - however clumsily.
On Monday, Issa and Jordan raised the stakes. They asked George to examine the activities of the IRS's Review of Operations Unit, which is a division of the same group involved in the original targeting controversy. A statement from the GOP side of the committee said the issue involved "allegations of political targeting."
Monday's letter asked George to determine whether the IRS singled out tax-exempt groups for additional review based on their political ideologies and whether the agency automatically flagged tax-exempt tea party groups.
The letter cites congressional interviews with IRS employees to show that the IRS examined right-leaning groups that had already been approved for tax-exempt status. It does not provide conclusive evidence that the agency applied scrutiny only to conservative organizations.
Rep. Elijah Cummings of Maryland, the oversight panel's ranking Democrat, accused Issa in a letter Monday of using selective quotes from the congressional interviews to make "unsubstantiated allegations."
"The committee has identified no evidence that the IRS discriminated against conservative groups that had been approved for tax-exempt status," Cummings said.
Cummings produced quotes of his own to show that IRS agents sometimes referred groups to the Review of Operations Unit in order to give them a chance while still keeping an eye on those organizations.
"We'll give them the benefit of the doubt, but we will try to take a look later without burdening them to just make sure that that was the right decision," an IRS employee said, according to the partial transcript.
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