Thirty members of the Actinomycetes family were screened for the ability to grow on various organic compounds as sole carbon sources. From this screen, Rhodococcus sp. 19070 was selected for further investigation. A 2.3 kb EcoRI restriction fragment was cloned from Rhodococcus sp. 19070 that hybridized to a fragment from the Pseudomonas putida mt-2 TOL plasmid, pWW0. This fragment from the TOL plasmid contained genes used in the catabolism of toluene. The nucleotide sequence of the 2.3 kb EcoRI fragment was determined and found to have sequence homology to the xylXYZ genes from the P. putida TOL plasmid and the benABC genes from Acinetobacter calcoaceticus. These two sets of genes encode benzoate dioxygenase, an enzyme used in the metabolism of toluene and benzoic acid. The amino acid identity among the three sets of genes was 51% for the first subunit, 47% for the second subunit, and 38% for the third subunit. The regions flanking the 2.3 kb EcoRI fragment were cloned on two BglII restriction fragments, 7.4 kb and 9.0 kb in size. A BamHI-EcoRI restriction fragment from the smaller clone was used to extend the 2.3 kb EcoRI fragment by an additional 0.9 kb. The nucleotide sequence of the entire 3.2 kb of the clone was determined and found to contain three complete open reading frames, which were named bopXYZ. Protein expression from bopXYZ showed three distinct proteins of 51.5-, 18.6-, and 37.5-kilodaltons on an SDS-PAGE gel. Biochemical assays of Escherichia coli cells expressing these three genes demonstrated benzoate transformation activity of the bopXYZ proteins