Mycobacterium vanbaalenii PYR-1 is able to metabolize a wide range of low- and high-molecular-weight (HMW) polycyclic aromatic hydrocarbons (PAHs). A 20-kDa protein was upregulated in PAH-metabolizing M. vanbaalenii PYR-1 cells compared to control cultures. The differentially expressed protein was identified as a beta subunit of the terminal dioxygenase using mass spectrometry. PCR with degenerate primers designed based on de novo sequenced peptides and a series of plaque hybridizations were done to screen the M. vanbaalenii PYR-1 genomic library. The genes, designated nidA3B3, encoding the alpha and beta subunits of terminal dioxygenase, were subsequently cloned and sequenced. The deduced enzyme revealed close similarities to the corresponding PAH ring-hydroxylating dioxygenases from Mycobacterium and Rhodococcus spp. but had the highest similarity, 61.9%, to the alpha subunit from Nocardioides sp. strain KP7. The alpha subunit also showed 52% sequence homology with the previously reported NidA from M. vanbaalenii PYR-1. The genes nidA3B3 were subcloned into the expression vector pET-17b, and the enzyme activity in Escherichia coli cells was reconstituted through coexpression with the ferredoxin (PhdC) and ferredoxin reductase (PhdD) genes of the phenanthrene dioxygenase from Nocardioides sp. strain KP7. The recombinant PAH dioxygenase appeared to favor the HMW PAH substrates fluoranthene, pyrene, and phenanthrene. Several other PAHs, including naphthalene, anthracene, and benz[a]anthracene, were also converted to their corresponding cis-dihydrodiols. The recombinant E. coli, however, did not show any dioxygenation activity for phthalate and biphenyl. The upregulation of nidA3B3 in M. vanbaalenii PYR-1 induced by PAHs was confirmed by reverse transcription-PCR analysis.