A novel benz[a]anthracene and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-inducible cytochrome P-450 (P450EF), which is very active in polycyclic aromatic hydrocarbon metabolism, has been purified from C3H10T1/2 mouse embryo fibroblasts (Pottenger, L. H., Christou, M., and Jefcoate, C. R. (1991) Arch. Biochem. Biophys. 286, 488-497). P450EF was shown to be immunologically unrelated to the major known P-450 families. A 4.9-kilobase (kb) cDNA encoding P450EF has been isolated from a lambda ZAP cDNA expression library generated from mRNA of TCDD-induced C3H10T1/2 cells. This cDNA comprises 175-base pair (bp) 5'-noncoding, 1629-bp open reading, and about 3100-bp 3'-noncoding sequence. A SmaI fragment of the 4.9-kb cDNA hybridized to a 5.2-kb mRNA species equally induced by benz[a]anthracene (10 microM) and TCDD (10 nM) in C3H10T1/2 cells, consistent with the involvement of the Ah receptor in this induction process. The deduced amino acid sequence (543 amino acids), the longest of any known cytochrome P-450, exhibits 41 and 38% identity to mouse CYP1A1 and CYP1A2, respectively, and less but substantial similarity (30-33% identity) to many members of the CYP2 family. There are five extended regions of > or = 50% identity to CYP1A1 as follows: (a) 51-118; (b) 199-222; (c) 326-343 (I-helix, O2-binding threonine); (d) 357-430; and (e) 460-487 (heme-binding cysteine). These sequence relationships suggest that P450EF is a member of a new CYP1B subfamily (mouse CYP1B1). Hybridization of mRNA and immunoblot analyses of microsomes both demonstrated beta-naphthoflavone (beta-NF) inducibility of Cyp1b-1 expression in C3H mouse lung, liver, and uterus although at lower levels relative to Cyp1a-1. The mobility of the beta-NF-inducible immunoreactive liver protein was significantly higher than that of the CYP1B1 protein detected in mouse lung, uterus, and C3H10T1/2 cells. Compared with the beta-NF-induced uterus, polycyclic aromatic hydrocarbon-induced uterine fibroblasts exhibited 10-20-fold higher levels of CYP1B1, suggesting that stromal fibroblasts are a major source of the protein.