Human cytochrome P450 1B1 (CYP1B1) was first isolated by differential hybridization as a 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-responsive cDNA clone from a human keratinocyte cell line treated with TCDD. Analysis of the complete cDNA sequence of this mRNA identified a new gene subfamily of cytochrome P450, CYP1B 1, based on 40% sequence homology to other polycyclic aromatic hydrocarbon (PAH)-inducible isoforms, CYP1A1 and CYP1A2. Initial characterization of the human CYP1B1 gene described the DNA sequence of a 12-kb genomic clone corresponding to the entire 5.1-kb CYP1B1 cDNA and containing 3.0 kb of upstream DNA. Comparison of these sequences revealed the location of three exons (371, 1044, and 3707 bp) and two introns (390 and 3032 bp), with the CYP1B 1 open reading frame spanning exons 2 and 3. High-resolution chromosome mapping confirmed the previous somatic cell hybrid analysis and placed the CYP1B1 gene at 2p21-22 of human chromosome 2. Comparison of the human CYPIB 1 genomic and cDNA sequences, obtained independently from two cell lines derived from different individuals, revealed three sequence differences, including an amino acid change at valine-432. Concurrent human genetic studies to identify one of two loci for primary congenital glaucoma (PCG) led to the mapping of the PCG locus GCL3A to human chromosome 2 at the 2p21 region. Subsequent investigations have led to independent reports that identify distinct CYP1B 1 gene mutations that segregate with the GCL3A phenotype in PCG families.