Previously, levels of a novel human mRNA, detected by a recombinant cDNA designated clone 1, were shown to be increased 50-fold in response to treatment of a keratinocyte cell line with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), in part as a function of increased rates of gene transcription (Sutter, T.R., Guzman, K., Dold, K.M., and Greenlee, W.F. (1991) Science 254, 415-418). Here we report the complete corresponding 5.1-kilobase cDNA sequence. A single open reading frame that predicts a protein of 543 amino acid residues was determined by computer-assisted analysis of the cDNA sequence. This predicted protein identifies a new gene subfamily of cytochrome P450, cytochrome P4501B1 (CYP1B1), that maps to human chromosome 2. Southern blot analysis of genomic DNA indicates that the human CYP1B subfamily is likely to contain only this single gene. Northern blot analysis of RNA isolated from primary cultures of normal human epidermal keratinocytes showed approximately 100-fold increased levels of the CYP1B1 mRNA after treatment with 10 nM TCDD for 24 h. Low levels of constitutive CYP1B1 mRNA were detected in 15 different human tissue samples. These results indicate that CYP1B1 is expressed in many normal human tissues and advance our understanding of the complexity of a gene family of cytochromes P450 whose expression is altered by TCDD.