A pyrene degrading bacterium, identified as Mycobacterium sp. strain S65, was isolated from a jet-fuel contaminated site in Quebec, Canada. Strain S65 utilized pyrene, phenanthrene, and fluoranthene as sole carbon and energy sources, but did not mineralize naphthalene, anthracene, or fluorene. Pyrene mineralization was enhanced by adding benz[a]anthracene, benzo[a]pyrene, or phenanthrene as co-substrates. Southern hybridization using the naphthalene inducible pyrene dioxygenase gene (nidA), from Mycobacterium vanbaalenii strain PYR-1, indicated that nidA homologues were found in two separate loci in strain S65. Each locus encoded two large subunit ring-hydroxylating dioxygenase genes (designated pdoA/X, and nidA/X), two alcohol dehydrogenase genes (designated pdoC/H and nidC/H) and one unknown orf (orfP6 and orfN4). The pdo locus also includes pdoB, which was predicted to function as a small subunit ring-hydroxylating dioxygenase gene. RT-PCR analyses showed that both nidA homologues, which were 99% and 89% identical to nidA from PYR-1, as well as the other genes in the two clusters, were induced during growth on pyrene and phenanthrene but not on glucose as sole carbon and energy source. The pdo locus also encoded for an IS3 like transposase upstream of pdoB. This transposase has a stop codon within its coding sequence indicating that it has lost functionality. Its' presence, however, suggests that there was a duplication of pyrene degrading genes within the genome. The presence of two separate pyrene degradation gene clusters in strain S65 may provide opportunities to explore how bacteria develop the abilities to degrade high-molecular-weight polycyclic aromatic hydrocarbons.