Mycobacterium sp strain CH-2 was isolated from a manufactured gas plant contaminated with polycyclic aromatic hydrocarbons (PAHs) and was identified by analysis of 16S rDNA sequences. Strain CH-2 was capable of mineralizing 3- and 4- ring PAHs, including phenanthrene, pyrene, and fluoranthene. In addition, strain CH-2 could utilize phenanthrene, pyrene and a wide range of alkanes as a sole carbon and energy source. Primers based upon the sequences of the polycyclic aromatic hydrocarbon (PAH) dioxygenases nidAB (from Mycobacterium vanbaalenii strain PYR-1) and pdoA2B2 (from Mycobacterium sp. Strain 6PY1) were used as molecular probes to amplify the dioxygenases. Degenerate primers were used to amplify a portion of an alkane monooxygenase gene. Mineralization assays and reverse transcriptase-polymerase chain reaction (RT-PCR) analysis indicated that the alkane monooxygenase was constitutively expressed, while nidAB and pdoA2B2 were expressed only in the presence of PAHs. A genomic library of strain CH-2 was created and then screened for the presence of biodegradative operons using the amplified PAH dioxygenases. The pdolocus included a partial pdoF, as well as pdoA2, pdoB2, orf 72, and putative genes for a ferredoxin, an araC-type regulator, and a reductase. The nid locus included a partial nidC, as well as nidB, nidA, and a putative promoter. Primer extension analysis of the nidlocus located the transcriptional start site 68bp upstream of the nidB start codon. The putatively identified promoter region and a promoter fragment lacking the -10 region were amplified, and the products were cloned into pRW50. This plasmid carries the lac operon without a promoter. The plasmid containing the full length promoter expressed the lacZ reporter gene, while expression by the promoter fragment was equivalent to the expression of cells carrying pRW50.