The covalent binding of acetaminophen (APAP) to mitochondrial proteins has been postulated to alter the function of the organelle and contribute to the development of the hepatotoxicity upon APAP overdose. To identify the arylated proteins CD-1 mice were administered 600 mg/kg APAP and Western blots of mitochondrial proteins collected 4 hr after dosing were probed with anti-APAP antibodies. Five proteins of approximately 75, 60, 54, 44, and 33 kDa were detected on 1-D gels. Immunostaining of the 54-kDa protein was most intense. Mitochondria were subsequently fractionated into inner and outer membrane, matrix, and intermembrane space using digitonin, sonication, and differential centrifugation. The 54-kDa target was most highly enriched in the inner membrane fraction. On 2-D gels this 54-kDa band was resolved into three arylated proteins with pIs of 6.4, 6.6, and 7.1. The pI 7.1 protein was excised from 55 2-D gels, and, after tryptic digestion, the two best-resolved peptides were sequenced and found to be 100% identical to mitochondrial aldehyde dehydrogenase. Coincident with APAP covalent binding the specific activity of the enzyme decreased; by the time of maximal covalent binding at 4 hr after APAP, the activity was 60% of control. Since the enzyme is an abundant mitochondrial dehydrogenase, its decreased activity may contribute to the impaired mitochondrial function observed after APAP administration.