Mycobacterium sp. PYR-1 was previously isolated in our laboratory and was shown to be able to mineralize high molecular mass polycyclic aromatic hydrocarbons (PAHs) [Heitkamp and Cerniglia, (1988) Appl. Environ. Microbiol. 54, 1612–1614]. In this research, the 16S rRNA gene (rDNA) of this strain was amplified by polymerase chain reaction (PCR) and directly sequenced by cycle sequencing method. We compared this sequence with all known mycobacterial 16S rDNA sequences available from GenBank and found that Mycobacterium sp. PYR-1 16S rDNA differs from the other mycobacteria, especially in the region of nucleotides 168–200 (in the Escherichia coli numbering system). Using the 16S rDNA sequences of the mycobacteria, a phylogenetic tree was constructed. The data from the phylogenetic tree and similarity values suggest that Mycobacterium sp. PYR-1 is closer to M. aurum and M. vaccae. Using the same approach, we also determined the 16S rDNA from an another PAH-degrading Mycobacterium sp. PAH135, isolated by Grosser and colleagues (1991) (Appl. Environ. Microbiol. 57, 3462–3469). Mycobacterium sp. PAH135 was found to be closer to M. aichiense, and different from our Mycobacterium sp. PYR-1.