A cytochrome P450 cDNA, encoding a new form of CYP3A protein, was isolated from a liver cDNA library of a male rat using anti-P450(6)beta-1 and anti-P450(6)beta-2 antibodies and the CYP3A2 cDNA. The cDNA (CYP3A18 cDNA) consisted of 1987 nucleotides, in which were contained an open reading frame of 1491 bp (corresponding to 497 amino acids), 5'-(59 bp) and 3'-noncoding regions (437 bp). The deduced amino acid sequence of CYP3A18 cDNA was completely identical in the first 27 N-terminal residues of P450(6)beta-2 previously isolated by us (Nagata et al. J Biochem 1990: 107, 718-725) from livers of rats treated with dexamethasone, and also shared higher extents of similarity with hamster CYP3A10 (78.5%) than with rat CYP3As previously sequenced (66.3-69.3%). Northern blot analyses indicated a male-dominant expression of this new CYP3A mRNA and enhanced expression in dexamethasone-or pregnenolone-16 alpha-carbonitrile (PCN)-treated, but not phenobarbital-or 3-methylcholanthrene-treated rats. Expressed CYP3A18 protein in COS-1 cells migrated at a position identical to that of purified P450(6)beta-2 on sodium dodecyl sulfate-acrylamide gel electrophoresis and catalyzed 16 beta- and 6 alpha-hydroxylations of testosterone. In contrast to CYP3A1 and CYP3A2, cytochrome b5 was not essential for maximal catalytic activities of recombinant CYP3A18 protein. These results, together with ontogenic profiles of CYP3A18 mRNA and P450(6)beta-2 protein, indicate that the newly isolated CYP3A18 cDNA encodes P450(6)beta-2 in rat liver.