Cytochrome P-450 3b has been shown previously to exist in one of two catalytically and structurally distinct forms. Preparations of P-450 3b obtained from outbred New Zealand White rabbits exhibit both high-efficiency (Vmax/Km) progesterone 6 beta- and 16 alpha-hydroxylases and a low-efficiency 16 alpha-hydroxylase. In contrast, P-450 3b prepared from a genetically defined strain of rabbits, IIIVO/J, does not display the high-efficiency 6 beta- and 16 alpha-hydroxylase activities. This appears to reflect the inheritance of one of two catalytically distinct subforms of P-450 3b. In the present study, we provide information with respect to the characterization of these progesterone hydroxylases as they occur in two highly inbred strains of rabbits, B/J and III/J. The identification of these strains as sources of these subforms was achieved by utilizing a monoclonal antibody to P-450 3b that produced a distinct pattern of inhibition for each phenotype. The functional and structural attributes of these subforms of P-450 3b indicate that whereas only the low-efficiency 16 alpha-hydroxylase is expressed in B/J rabbits, both of the catalytically distinct subforms of P-450 3b are expressed in the III/J rabbits. Thus, it is unlikely that these enzymic variants reflect allomorphic forms of P-450 3b.