Polyclonal antibody against a cytochrome P-450 expressed in adult male rats was utilized to screen a lambda gt-11 library. Two cDNAs were isolated and sequenced. The cDNA and deduced protein sequence revealed that these P-450s correspond to previously isolated P-450 PB1 (Waxman D. T., Ko, A., and Walsh, C., (1983) J. Biol. Chem. 258, 11937-11947) and P-450f (Ryan, D. E., Iida, S., Wood, A. W., Thomas, P. E., Lieber, C. S., and Levin, W., (1984) J. Biol. Chem. 259, 1239-1250). Both P-450s contain 490 amino acids and share 75% similarity with each other and 50% similarity with P-450b and P-450e. Southern blot analysis indicates that these enzymes are part of a small P-450 gene subfamily composed of two or three members. An unusual feature of P-450 PB1 is the presence of a rat brain identifier-type repetitive sequence (R.dre.1) that encompasses the complete 3' untranslated region of the mRNA. This sequence is absent from P-450f mRNA. A cDNA from an apparent allelic form of P-450 PB1 mRNA was isolated and sequenced. This cDNA was identical to the other P-450 PB1 cDNA except for a single base substitution in the R.dre.1 repeat and the presence of a repeat (AAATAAA)13 at the 3' end of the mRNA. By use of a 3' specific probe for P-450f mRNA and a probe common to P-450 PB1 and P-450f mRNAs, the mechanism by which these P-450s are elevated during rat development was studied. Both RNAs are virtually absent in newborn rats and become elevated between 1 and 4 weeks after birth. Maximal levels of P-450f mRNA levels continue to increase up to 12 weeks after birth, and 2-fold higher levels of P-450f mRNA are reached in 12-week-old female rats compared with male rats. P-450 PB1 mRNA reached maximal levels at 4 weeks and no sex difference in mRNA level was detected for P-450 PB1. Nuclear run-on transcription assays confirmed that the increase in these mRNAs is due to transcriptional activation.