Debrisoquine 4-hydroxylase (P450db1) was purified from rat liver microsomes. Polyclonal antibody was produced and, in conjunction with immunoblots, was used to identify and purify a second immunorelated P450 (P450db2) that does not have debrisoquine hydroxylating activity. The cDNA clones to db1 and db2 were isolated from a lambda gt11 expression library, sequenced, and found to share 78% nucleotide and 73% deduced amino acid similarities. These similarities are evenly dispersed along the sequence except for a region of 190 nucleotides with 99% similarity near the carboxyl terminus of the protein-coding region; this similarity is probably the remnant of a gene conversion event. Both proteins share between 38% and 43% amino acid similarity with P450a, P450b, P450e, P450f, P450PB1, and P450j; these data indicate that P450db1 and P450db2 are members of a separate subfamily within the P450II gene family. Southern blot analysis and preliminary genomic cloning suggest that at least four genes exist in the subfamily, although the present evidence suggests that only db1 and db2 are expressed in rat liver. With the use of 19 mouse X hamster somatic cell hybrids, the db1 and db2 genes were localized to mouse chromosome 15 (P450-2D locus). A polymorphism has been described for debrisoquine metabolism in the DA rat strain, adult females having markedly decreased debrisoquine 4-hydroxylase activity. Our immunoblot analysis and mRNA analysis suggest that debrisoquine 4-hydroxylase deficiency in the female DA rat is not due to a decrease in db1 protein or mRNA. The db1 and db2 proteins are differentially regulated: during development db2 is present at birth while db1 is absent, and db1 increases by 1 week of age; in addition, db1 is slightly induced by phenobarbital, 3-methyl-cholanthrene, and dexamethasone whereas db2 is marginally increased by these latter two agents. These results demonstrate that debrisoquine 4-hydroxylase is a member of a new constitutively expressed P450II sub-family containing two or more genes in the rat and establish that the debrisoquine polymorphism in the DA rat is probably due to a structurally altered db1 protein.