Previously, we have shown that highly specific antibodies against cytochrome P450 enzymes can be produced by targeting a 5-amino acid sequence at the C-terminus. Although rat CYP3A1 and CYP3A2 share 89% amino acid sequence similarity, they differ by 3 out of 5 of their C-terminal residues. In an effort to produce antibodies specific to each form, rabbits were immunised with the peptides IITGS and VINGA, corresponding to the C-termini of CYP3A1 and CYP3A2, respectively. Both antibodies bound strongly to hepatic microsomal fraction from rats treated with pregnenolone 16 alpha-carbonitrile (PCN) in enzyme-linked immunosorbent assay. Binding of the anti-IITGS antibody was strongly inhibited by incubation with IITGS, but VINGA was 60 times less effective. Conversely, binding of the anti-VINGA antibody was inhibited by VINGA 100 times more effectively than IITGS. Similar inhibition of antibody binding was also found using immunoblotting. Immunoadsorption using the anti-IITGS antibody yielded a single protein from solubilised hepatic microsomal fraction from PCN-treated rats, which was recognised only by the anti-IITGS antibody. Both antibodies bound to single proteins in the liver which were increased following treatment with PCN, but only the anti-IITGS antibody recognised protein in the lung, small intestine, and kidney of untreated and PCN-treated rats. Also, the binding of the two antibodies to hepatic and extrahepatic microsomal fractions from uninduced and induced rats showed differences in the expression of proteins recognised by the two antibodies, providing further evidence of antibody specificity. Thus, the binding of anti-IITGS and anti-VINGA antibodies is mutually exclusive and consistent with specific binding to their target antigens, CYP3A1 and CYP3A2, respectively. Immunocytochemistry was used to determine the distribution of CYP3A1 and CYP3A2. In the liver of untreated animals, both CYP3A1 and CYP3A2 were found to be expressed in the centrilobular region. However, some CYP3A1 immunoreactivity was also detected in many, but not all, hepatocytes throughout the lobule. However, following treatment of rats with PCN, both CYP3A1 and CYP3A2 were found to be strongly expressed in hepatocytes throughout the lobule, although CYP3A2 showed greater expression in the centrilobular region. PCN treatment was also found to result in induction of CYP3A1 in specific regions of the small intestine, lung, and kidney.