A cytochrome P450 cDNA clone, designated pP45OPCN2, homologous to the previously characterized pregnenolone 16a-carbonitrile (PCN)-induced P450 cDNA (pP450PCN1; F. J. Gonzalez, D. W. Nebert, J. P.Hardwick, and C. B. Kasper, J. , was isolated from a rat liver cDNA expression library by use of a polyclonal anti-P45OPCNl antibody. This P-450 cDNA contains 2,014 base pairs and yields an open reading frame of a protein consisting of 504 amino acids (Mr = 57,760). P45OPCN2 cDNA and protein shared 90% nucleotide and 89% amino acid imilaity with P45OPCN1 cDNA and protein, respectively. The 5' untranslated, coding, and 3' untranslated regions between the two cDNAs share 94, 93, and 79% similarities, respectively. Nucleotide differences in the coding regions, however, are not evenly distributed. Complete homology exists between the two mRNAs for 425 nucleotides (positions 346 through 771). Other regions of 93 nucleotides containing only one difference and 147 nucleotides containing two differences exist toward the 3' end of the coding regions. These data suggest the possibility that a gene conversion event(s) have occurred subsequent to duplication of the ancestral P45OPCN gene. Oligonucleotide probes unique for P45OPCN1 and P45OPCN2 cDNAs were used to examine the levels of their respective mRNAs in noninduced and PCN-induced liver cells and in male and female rats of various ages. P45OPCN1 mRNA was not detectable in either male or female rats at any ages. In contrast, P45OPCN2 mRNA was present at a low level in newborn rats and became elevated in both males and females at 1 week of age. Levels of P45OPCN2 mRNA continued to increase in males until 12 weeks, whereas the mRNA in females reached peak levels at 2 weeks of age but declined continuously at the onset of puberty (between 4 and 12 weeks). These levels of P45OPCN2 mRNA clsely parallel the increases in testosterone 63-hydroxylase activity and P45OPCN2 protein level, as analyzed by Western blots. P450PCN1 mRNA was induced by PCN, dexamethasone, and phenobarbital in both male and female rats. P45OPCN2 mRNA was not sgnilicntiy induced by PCN or dexamethasone but was readily induced by phenobarbital. Testosterone 6P-hydroxylase activity was also induced severalfold by PCN, dexamethasone, and phenobarbital. These data demonstrate that P45OPCN1 and P45OPCN2 genes are differentially regulated during development and after administation of inducing compounds and furthermore suggest that both enzymes possess testosterone 6I-hydroxylase activity.The primary components of the hepatic microsomal multisubstrate monooxygenase system are NADPH-cytochrome P-450 oxidoreductase and cytochrome P-450 (P-450). The former is a single molecular species that transfers reducing equivalents from NADPH to P-450. P-450s are a population of related enzymes that range in size from 49,000 to 58,000 daltons and contain noncovalently bound heme. In some cases, foreign compounds and endogenous substances regulate levels of specific P-450 species (22, 47). The structure (1, 5a), regulation (1,30,46), ...