Anti-liver/kidney microsome1-positive sera from children with chronic active hepatitis were studied in an effort to identify the microsomal antigens selected during induction and progression of this autoimmune disease. Immunoblot analysis of sodium dodecyl sulfate gel-resolved microsomal proteins from human and rat liver using anti-liver/kidney microsome1-positive sera revealed a single polypeptide of 48 kilodaltons (human microsomes) or 50 kilodaltons (rat microsomes). Levels of the 50-kilodalton rat microsomal polypeptide were suppressed in vivo by several drugs known to modulate expression of individual forms (enzymes) of hepatic cytochrome P-450, with the largest decrease effected by phenobarbital. Dot blot analysis using a panel of 10 electrophoretically homogeneous rat liver cytochrome P-450 forms under nondenaturing conditions established that the two methylcholanthrene-inducible forms, P-450 BNF-B and P-450 ISF-G (P-450 gene subfamily IA), are selectively recognized by the anti-liver/kidney microsome1 antibodies. These findings demonstrate that sera associated with autoimmune (anti-liver/kidney microsome1) chronic active hepatitis are specifically reactive with select rat hepatic P-450 forms and suggest that these autoantibodies may be principally directed against one or more constitutive forms of the corresponding human liver cytochromes.