Previous work has shown that P-450 2B11 is responsible for the unique ability of dogs to metabolize and eliminate certain highly-chlorinated biphenyls such as 2,2',4,4',5,5'-hexachlorobiphenyl (245-HCB), whereas the related P-450 2B forms in rat and rabbit are unable to metabolize the compound to any significant degree. To determine the structural basis for this functional diversity, hybrid enzymes were generated. Success with this approach required a careful choice of second enzyme and common substrate with which to assess the functional integrity of the hybrid proteins. The choices of P-450 2B5 from rabbit as the second enzyme and androstenedione as the substrate were based in part on the finding that P-450 2B11 and P-450 2B5 hydroxylate androstenedione with similar overall activities but distinct profiles. Enzymatic studies with eight hybrid enzymes provided evidence for two regions of P-450 2B11 and 2B5, between residues 95-239 and 240-370, that appear to be involved in defining substrate specificity for androstenedione, and three regions of P-450 2B11, between residues 95-239, 240-370, and 371-494, that contain amino acids necessary for metabolism of 245-HCB. This deliberate approach to the creation of hybrid cytochromes P-450 has generated a series of enzymes that will be central to further structure-function studies of the cytochromes P-450 2B.