Rhodococcus opacus R7 was isolated from a soil contaminated with polycyclic aromatic hydrocarbons for its ability to grow on naphthalene. The strain was also able to degrade o-xylene, the isomer of xylenes most recalcitrant to microbial degradation. The catabolic pathways for naphthalene and o-xylene were investigated by identification of metabolites in R. opacus R7 cultures performed with the two hydrocarbons and by evaluation of some enzymes involved in the metabolism of these compounds. 1,2-Dihydro-1,2-dihydroxynaphthalene, salicylic and gentisic acids were identified as metabolites in cultures exposed to naphthalene. This suggests that the degradation occurs through the dioxygenation of the aromatic ring with the formation of 1,2-dihydro-1,2-dihydroxynaphthalene, dehydrogenated to the corresponding 1,2-dihydroxy derivative which is further oxidized to salicylic acid, a key intermediate of naphthalene metabolism; this compound is converted to gentisic acid cleaved by a gentisate 1,2-dioxygenase. From R. opacus R7 cultures supplied with o-xylene, 2,3-dimethylphenol and 3,4-dimethylcatechol were observed. The pathway of o-xylene involves the monooxygenation of the benzene nucleus leading to dimethylphenol which is further metabolised to 3,4-dimethylcatechol, followed by a meta cleavage reaction, catalyzed by the catechol 2,3-dioxygenase. R. opacus R7 is the first strain thus far described both in Gram-negative and Gram-positive bacteria which has the ability to degrade both a polycyclic aromatic hydrocarbon such as naphthalene and a monocyclic aromatic hydrocarbon such as o-xylene.