The entire nucleotide sequences (6.8 kilobase pairs) of the bphABC genes and their products involved in the initial dioxygenation and ring-meta-cleavage of biphenyls and polychlorinated biphenyls were determined. The first bphA gene starts about a 100 base pairs downstream from the transcriptional initiation site. The bphA region, which encodes a cluster of enzymes including biphenyl dioxygenase catalyzing the initial catabolic step, consists of five open reading frames (ORFs). Five proteins corresponding to these ORFs in the molecular masses were detected by in vitro protein synthesis, of which four ORFs are very similar to the recently reported todC1C2BA genes coding for the corresponding enzymes catalyzing the initial dioxygenation reactions of toluene (Zylstra, G.J., and Gibson, D. T. (1989) J. Biol. Chem. 264, 14940-14946). The third open reading frame (ORF3) of the bphA region, missing its counterpart in the toluene dioxygenase gene cluster, was site-specifically deleted, and the resulting enzymatically active mutant reveals that this ORF3 is not mandatory for the catabolism of biphenyls. Thus the biphenyl degradation pathway and the responsible enzymes/genes are very similar to those of toluene degradation despite their discrete substrate specificity.