The monoclonal antibody, 3/4/2, which was raised against purified rat cytochrome P450 isoenzyme 1A1 (CYP1A1) binds to cytochromes P4501A in many species. It was shown by immunoblotting that the antibody binds to CYP1A1 in microsomal fractions prepared from rat, mouse, rabbit, hamster and human. The antibody also binds to cytochrome P450 isoenzyme 1A2 in microsomal fractions prepared from rabbit and human, but not rat or mouse. Using purified isoenzymes in an enzyme-linked immunosorbent assay it was found that the affinity of binding to the two rabbit hydrocarbon-inducible isoenzymes is reduced compared with that for rat CYP1A1. Binding is not affected by denaturation of the antigens. The effects of chemical and enzymatic treatments on rat CYP1A1 showed that the epitope contains a trypsin-sensitive site that includes arginine, but lacks lysine. The epitope does not contain methionine, cysteine, aspartic acid or glutamic acid residues. In addition, digestion of the protein with cyanogen bromide produces a fragment of Mr 20,000 which contains the antibody binding site. By comparing the cross-reactivity of the antibody with the primary structures of CYP1A1 and 1A2 from the rat, mouse, rabbit and human, and by considering the results of the chemical and enzymatic treatments, it was possible to deduce the likely location and structure of the binding site of 3/4/2 on members of the CYP1A subfamily. It is concluded that the epitope for this antibody is Phe-Arg-His-Ser-Ser-Phe, which lies at positions 380-385 in rat CYP1A1. Further, it is predicted from a model of the tertiary structure of eukaryotic cytochrome P450 that a part of this binding site lies within a helix in the native protein.