Mycobacterium vanbaalenii PYR-1 is capable of degrading polycyclic aromatic hydrocarbons (PAHs) to ring cleavage metabolites. This study identified and characterized a putative phthalate degradation operon in the M. vanbaalenii PYR-1 genome. A putative regulatory protein (phtR) was encoded divergently with five tandem genes: phthalate dioxygenase large subunit (phtAa), small subunit (phtAb), phthalate dihydrodiol dehydrogenase (phtB), phthalate dioxygenase ferredoxin subunit (phtAc) and phthalate dioxygenase ferredoxin reductase (phtAd). A 6.7 kb EcoRI fragment containing these genes was cloned into Escherichia coli and converted phthalate to 3,4-dihydroxyphthalate. Homologues to the operon region were detected in a number of PAH-degrading Mycobacterium spp. isolated from various geographical locations. The operon differs from those of other Gram-positive bacteria in both the placement and orientation of the regulatory gene. In addition, the M. vanbaalenii PYR-1 pht operon contains no decarboxylase gene and none was identified within a 37 kb region containing the operon. This study is the first report of a phthalate degradation operon in Mycobacterium spp.