Expression of several forms of cytochrome P450 2B and of P450 4B1 in rabbit kidney was investigated by cloning from cDNA libraries constructed with renal mRNA from animals treated with phenobarbital. Isolation and sequencing of several cDNAs demonstrated that: (i) cytochrome P450 2B-B0 can be found in rabbit kidney along with a newly discovered form of P450 2B termed "P450 2B-Bx." P450 2B-Bx differs from P450 2B-B0 at 25 nucleotide positions and at four positions in the derived sequence of 491 amino acids. Two previously identified forms of cytochrome P450 2B, 2B-B1 and 2B-B2, are not detected in rabbit kidney. cDNA encoding cytochrome P450 4B1 was also cloned from the kidney library and found to be identical in sequence to cDNAs cloned from rabbit hepatic and pulmonary libraries. Analysis of renal mRNA indicates that forms of cytochrome P450 2B and P450 4B1 are expressed in a number of species but induced by phenobarbital in rabbit only (4B1) or rabbit and hamster (2B). Relatively high levels of mRNA related to P450 4B1 were detected in samples from untreated and phenobarbital-treated mice. Analysis of protein by immunoblotting was less sensitive but produced results consistent with those obtained by analysis of mRNA; protein related to cytochrome P450 2B was detected in renal microsomal samples from rabbit and hamster (phenobarbital > untreated), and protein related to P450 4B1 in samples from rabbits (phenobarbital > untreated) and mice (phenobarbital = untreated). The four forms of cytochrome P450 2B were expressed in COS-7 cells, and their activities were evaluated with androstenedione, testosterone, and 7-ethoxycoumarin as substrates. Three of the P450 2B forms, B0, B1, and Bx, metabolize these substrates in a manner characterized by preference for 16 beta-hydroxylation of androstenedione, low testosterone 16-hydroxylation, and high ethoxycoumarin O-deethylation. The fourth form, P450 2B-B2, is catalytically distinct from the others, with activities characterized by high androstenedione 16 alpha- and 15 alpha-hydroxylation and high testosterone 16-hydroxylation. Since P450 2B-B2 is catalytically distinct from the other forms, the metabolic profiles of phenotypes that include P450 2B-B2 might differ significantly from those of phenotypes that lack P450 2B-B2.