It has been shown that large doses of acetaminophen can result in increased degradation of the hepatic cytochrome P450 (CYP) enzymes in vivo; however, the proteolytic pathways have not been identified. We found that incubating transfected HepG2 cells that express CYP3A4 or a reconstituted microsomal model containing human liver microsomes and cytosol, high concentrations of acetaminophen could induce a dose- and time-dependent degradation of CYP3A4. In the microsomal model the degradation could be blocked and augmented by the presence of catalase and superoxide dismutase, respectively. Tocopherol could also protect against the acetaminophen-induced degradation. However, lipid peroxidation assays showed no significant increases in lipid peroxidation products nor was there any protection by propyl gallate. Protease and proteasome inhibitors showed that the proteolytic process was mainly (85%) mediated by the lysosomal pathway, whereas a minor portion (15%) of the degradation was mediated by the proteasomal pathway. Both pepstatin A and anti-cathepsin D neutralizing antibody decreased acetaminophen-induced degradation of CYP3A4 in microsomal model systems. Pepstatin A also blocked the acetaminophen-induced degradation of the CYP3A4 in a transfected HepG2 cell line. Incubating the 3A4 cells in the presence of acetaminophen also increased cathepsin D content and activity. The lysosomal pathway, mainly mediated by cathepsin D, appears to be the major proteolytic pathway involved in the degradation of the P450 enzymes induced by toxic doses of acetaminophen.