The rat CYP2B gene subfamily includes CYP2B1, CYP2B2 and CYP2B3. Translation of an alternatively spliced hepatic CYP2B2 mRNA would generate a CYP2B2 variant, CYP2B2v, having eight additional amino acid residues inserted between CYP2B2 positions 274 and 275. The presence of CYP2B3 and CYP2B2v in rat liver has yet to be demonstrated. cDNA expression vectors were obtained for CYP2B1, CYP2B2, CYP2B3 and CYP2B2v. All four proteins react with an anti-CYP2B1 antibody and can be resolved by SDS-PAGE. A CYP2B3-specific polyclonal antibody raised against an undecapeptide (SPVDPNTIDMT) from near the C-terminus of CYP2B3 detected a constitutive protein on immunoblots of rat liver microsomes, thus demonstrating that the CYP2B3 mRNA is translated in the liver. Similarly, a CYP2B2v-specific polyclonal antibody was raised against a peptide containing the eight additional amino acid residues (VSPAWMRE) predicted to be present in the CYP2B2v protein. It detected a phenobarbital- and Aroclor 1254-inducible protein in rat liver microsomes. Microsomes of Ad293 cells expressing cDNAs for CYP2B2 and CYP2B2v were used to metabolize 7,12-dimethylbenz[a]anthracene (DMBA), and the metabolites produced were compared with those generated by microsomes of cells expressing CYP2B1 cDNA. CYP2B2v had activity similar to that of CYP2B2 for DMBA metabolism. Both CYP2B2 forms preferentially catalyzed 12-hydroxylation, whereas CYP2B1 preferred 7-hydroxylation and exhibited turnover that was strongly suppressed as previously reported. These results demonstrate the existence in rat liver of two new CYP2B proteins: CYP2B3, the major constitutive CYP2B form, and CYP2B2v, which represents a rare case of non-aberrant alternative splicing among xenobiotic-metabolizing P450s.