A key pathologic event in cardiac ischemia reperfusion (I-R) injury is mitochondrial energetic dysfunction, and several studies have attributed this to complex I (CxI) inhibition. In isolated perfused rat hearts, following I-R, we found that CxI-linked respiration was inhibited, but isolated CxI enzymatic activity was not. Using the mitochondrial thiol probe iodobutyl- triphenylphosphonium in conjunction with proteomic tools, thiol modifications were identified in several subunits of the matrix-facing 1α sub-complex of CxI. These thiol modifications were accompanied by enhanced ROS generation from CxI, but not complex III. Implications for the pathology of cardiac I-R injury are discussed. © 2005 Elsevier B.V. All rights reserved.