Two new bacterial strains, KOTLB and RN402, which have the ability to utilize pyrene as their sole source of carbon and energy, were isolated from soil. Strains KOTLB and RN402 degraded 99% of 100 mg/l of pyrene in liquid cultures within 16 days. Besides pyrene, both strains also degraded 100 mg/l of phenanthrene almost completely within 8 days and degraded 99% and 55% of 100 mg/l of fluoranthene within 24 days, respectively. 16S rDNA-based phylogenetic analysis suggested that strains KOTLB and RN402 were Diaphorobacter sp. and Pseudoxanthomonas sp., respectively. These two genera have never been reported to be involved in pyrene degradation. Polymerase chain reaction (PCR) amplification with nidA-specific primers revealed the presence of a dioxygenase gene in both strains. The partial nidA gene products of both strains (317 and 384 amino acids, respectively) exhibited 99-100% identity to the NidA proteins, which are the large subunit of terminal dioxygenases for pyrene degradation of Mycobacterium spp. In addition, PCR further demonstrated that nidA genes of these strains were located on a megaplasmid. This is the first report showing that pyrene-degrading Gram-negative bacteria harbor the nidA gene.