Autoantibodies against soluble liver enzymes have been reported among alcoholics, but the targets of self-reactivity toward membrane proteins of the liver have not been characterized. Previously, among alcoholics, we found antibodies against ethanol-derived radical protein adducts that are dependent on cytochrome P-4502E1 (CYP2E1) for their formation. To further investigate autoantibodies against cytochrome P-450s during alcohol abuse, sera of rats chronically treated with ethanol in the total enteral nutrition model and sera from alcoholics with or without alcohol liver disease and from control subjects were analyzed by enzyme-linked immunosorbent assay and Western blotting for the presence of IgG against rat and human CYP2E1, rat CYP3A1, and human CYP3A4. A time-dependent appearance of IgG against rat CYP3A1 and CYP2E1 was evident during chronic ethanol feeding of rats. Anti-CYP2E1 reactivity showed positive correlation with the levels of hepatic CYP2E1 and was inhibited by the CYP2E1 transcriptional inhibitor chlormethiazole. Screening of the human sera by enzyme-linked immunosorbent assay revealed reactivity against CYP3A4 and CYP2E1 in about 20 to 30% and 10 to 20% of the alcoholic sera, respectively. No difference were noted between sera from alcoholics with or without hepatitis C virus infection, and only very little reactivity was seen in sera from control subjects. Western blotting analysis revealed anti-human CYP2E1 reactivity in 8 of 85 alcoholic sera and 3 of 58 control sera, whereas anti-CYP3A4 reactivity was detected in 18 of 85 alcoholic sera and 4 of 58 control sera, which were different from the sera reactive with CYP2E1. Immunoblot reactivity of CYP3A4-positive alcoholic sera was found against glutathione-S-transferase fusion proteins containing truncated forms of CYP3A4, and such sera were also able to immunoprecipitate in vitro translated CYP3A4. Seven of eight sera showed reactivity toward domains C-terminal of position Ser281, and 1 of 8 sera recognized autoepitopes within the region Thr207-Ser281. These findings indicate that alcoholics develop autoantibodies against CYP2E1 and CYP3A4 that the CYP3A4 C-terminal domain is a target for the autoantibody reactions among a subset of alcoholics. The novel finding of CYP3A4 autoantibodies and their significant expression among alcoholics warrants further investigation. Attention should be given to immune toxicity associated with CYP3A4 autoantibodies and cases of alcohol abuse that are accompanied by exposure to drugs and substances that are CYP3A substrates.