The degradation of pyrene, a polycyclic aromatic hydrocarbon containing four aromatic rings, by pure cultures of a Mycobacterium sp. was studied. Over 60% of [14C]pyrene was mineralized to CO2 after 96 h of incubation at 24 degrees C. High-pressure liquid chromatography analyses showed the presence of one major and at least six other metabolites that accounted for 95% of the total organic-extractable 14C-labeled residues. Analyses by UV, infrared, mass, and nuclear magnetic resonance spectrometry and gas chromatography identified both pyrene cis- and trans-4,5-dihydrodiols and pyrenol as initial microbial ring-oxidation products of pyrene. The major metabolite, 4-phenanthroic acid, and 4-hydroxyperinaphthenone and cinnamic and phthalic acids were identified as ring fission products. 18O2 studies showed that the formation of cis- and trans-4,5-dihydrodiols were catalyzed by dioxygenase and monooxygenase enzymes, respectively. This is the first report of the chemical pathway for the microbial catabolism of pyrene.