Protein thiols with regulatory functions play a critical role in maintaining the homeostasis of the redox state in mitochondria. One major host of regulatory cysteines in mitochondria is Complex I, with the thiols primarily located on its 51 kDa FMN-binding subunit. In response to oxidative stress, these thiols are expected to form intramolecular disulfide bridges as one of their oxidative post-translational modifications. Here, to test this hypothesis and gain insights into the molecular pattern of disulfide in Complex I, the isolated bovine Complex I was prepared. Superoxide (O(2)(.-)) is generated by Complex I under the conditions of enzyme turnover. O(2)(.-)-induced intramolecular disulfide formation at the 51, kDa subunit was determined by tandem mass spectrometry and database searching, with the latter accomplished by adaptation of the in-house developed database search engine, MassMatrix [Xu, H., et al., J. Proteome Res. 2008, 7, 138-144]. LC/MS/MS analysis of tryptic/chymotryptic digests of the 51 kDa subunit from alkylated Complex I revealed that four specific cysteines (C(125), C(142), C(187), and C(206)) of the 51 kDa subunit were involved in the formation of mixed intramolecular disulfide linkages. In all, three cysteine pairs were observed: C(125)/C(142), C(187)/C(206), and C(142)/C(206). The formation of disulfide bond was subsequently inhibited by superoxide dismutase, indicating the involvement of O(2)(.-). These results elucidated by mass spectrometry indicate that the residues of C(125), C(142), C(187), and C(206) are the specific regulatory cysteines of Complex I and they participate in the oxidative modification with disulfide formation under the physiological or pathophysiological conditions of oxidative stress.