1. In order to obtain specific antibodies of the P4502D subfamily, we generated two anti-peptide antibodies against synthetic peptides, DPAQPPRD (peptide A) and DPTQPPRH (peptide B). The sequence of peptide A occurs in rat P4502D2, P4502D4 and human P4502D6, whereas the sequence of peptide B occurs in the dog P4502D subfamily. These sequences are closely related to an epitope of liver/kidney microsomal autoimmune hepatitis. 2. In immunoblotting studies, the anti-peptide antibody against peptide A recognized a 49-KDa protein in microsomes derived from human lymphoblasts expressing P4502D6 and rat liver. It showed no crossreactivity with microsomes from dog liver. In contrast, the anti-peptide antibody against peptide B recognized a 49-KDa protein only in microsomes of dog liver. These indicate that each anti-peptide antibody has the specificity for the respective sequences of the members of P4502D subfamily, with the species investigated herein. 3. In immunoinhibition studies, the anti-peptide antibodies against peptide B inhibited bunitrolol 4-hydroxylation and propranolol 4,5-hydroxylation, which are mediated by the dog P4502D subfamily. These data suggest that the anti-peptide antibodies against peptide B bind to the native and denatured forms of the P4502D subfamily. 4. The present study has demonstrated that the anti-peptide antibodies against this region are useful for studying the members of the P4502D subfamily.