P450/6 beta A gene was isolated from a rat genomic library and its encoding protein was characterized by the expression of the corresponding cDNA in COS-1 cells. Between exon regions of P450/6 beta A gene and P450PCN2 cDNA (F. J. Gonzalez, B.-J. Song, and J. P. Hardwick (1986) Mol. Cell. Biol. 6, 2969-2976), 12 nucleotide differences were observed, involving two amino acid changes, His-->Asp [429] and Asp-->Gly [445], respectively. A cDNA (6 beta-A cDNA), whose nucleotide sequence was completely identical with the corresponding exon of P450/6 beta A gene, was isolated from a rat cDNA library. Northern blotting using specific oligonucleotide probes showed that 6 beta-A mRNA, but not P450PCN2 mRNA, was a major form in livers of the male rats. 6 beta-A protein expressed in COS-1 cells (at about 0.1 to 0.3% of total microsomal protein) catalyzed testosterone 6 beta-hydroxylation. The reaction was enhanced by the addition of NADPH-P450 reductase (2.5-fold) and by the simultaneous addition of cytochrome b5 and NADPH-P450 reductase (13.7-fold). Catalytic properties of 6 beta-A for typical CYP3A substrates were consistent with those of purified rat P450(6 beta-1) and P450(6 beta-3), corresponding, respectively, to CYP3A2 or the variant form (K. Nagata, F. J. Gonzalez, Y. Yamazoe, and R. Kato (1990) J. Biochem. 107, 718-725). Apparent Michaelis constants (Km) of 6 beta-A for testosterone 6 beta-hydroxylation and a O-dealkylation ratio of propoxycoumarin to pentoxycoumarin, however, showed higher degrees of similarity to those of purified P450(6 beta-3) than those of P450(6 beta-1).