Cytochrome P-450-mediated responses to environmental challenges are well known in diverse animal taxa, but the evolution of the complex gene superfamily coding for these enzymes is poorly understood. Here we report a phylogenetic analysis of the cytochrome P-450 1A (CYP1A) genes including two new sequences determined from teleost fish, toadfish (Opsanus tau) and scup (Stenotomus chrysops). Degenerate PCR primers were used to amplify a 1.2 kbp fragment from liver cDNA. The toadfish PCR product was used as a probe to identify a full-length CYP1A clone from a toadfish liver cDNA library. The entire coding region of the scup CYP1A was obtained by rapid amplification of cDNA ends (RACE) using specific primers based on the sequence of the partial PCR product. The predicted protein sequences for toadfish and scup CYP1A shared 78% and 83% amino acid identity with rainbow trout CYP1A1 respectively. Amino acid identity with mammalian CYP1A proteins ranged from 51 to 60% for 505 aligned positions. Phylogenetic analysis of four teleost fish CYP1A genes (trout, toadfish, scup and plaice) and 12 mammalian CYP1A genes suggests a monophyletic origin of the teleost genes, with the trout gene being most divergent, and indicates three distinct groupings: mammalian 1A1, mammalian 1A2, and fish 1A. This supports the idea that the gene duplication event which gave rise to CYP1A1 and CYP1A2 occurred after the divergence of the lines leading to mammals and fish. These results establish a molecular phylogeny within the CYP1A subfamily, the first such detailed phylogenetic analysis within a cytochrome P-450 family.