Coumarin 7-hydroxylase (P450coh) and steroid 15a-hydroxylase (P45015a) are encoded by members within the mouse 2A subfamily. Since P450coh activity is regulated by the Coh locus, we characterized P450coh cDNAs in strains having high coumarin 7-hydroxylase activity (CohHhomozygote) including 129/J and DBA/2J, and compared them with P450coh cDNAs in low activity strains (CohL homozygote) C57BL/6J, C3H/HeJ and AKR/J. The nucleotide sequences of these two cDNAs differ by a single base, which results in an amino acid difference at position 117 (Val in P450cohHand Ala in P450cohL). The CohHphenotype exhibits approximately 10-fold greater Vmaxand four-fold lower Km values than those in the CohL. Male 129AKF1/J expresses approximately equal amounts of P450cohHand P450cohLmRNAs, associated with two Coh alleles. The levels of P450coh and P45015xmRNAs in the F1offspring suggested that a trans -acting factor(s) appeared to regulate the expressions of the P450 genes. A recent duplication in the ancestral mouse established the line of descent to P45015xfrom the ancestral P450coh gene. During evolution, amino acid substitutions have selectively occurred at positions which alter the enzyme's substrate specificity and increase in the specific activity. Consistent with an important role of natural selection in the evolution of these genes is the relatively high nonsynonomous substitution rates on the P45O15a and the P450coh branches. As a result of these evolution events, the gene family consists of members which exhibit an extremely high degree of structural similarity, but very divergent hydroxylase activities and modes of regulation.