Rhodococcus sp. strain DK17 is able to utilize a variety of monocyclic aromatic hydrocarbons, including benzene, phenol, toluene, and o-xylene, as growth substrates. Although DK17 is unable to grow on m- and p-xylene, this strain could transform these two xylene isomers to some extent after induction by o-xylene. The major accumulating compounds formed during the degradation of m- and p-xylene by DK17 were isolated by high-pressure liquid chromatography and identified by gas chromatography-mass spectrometric and (1)H nuclear magnetic resonance spectral techniques. Both xylene isomers were transformed to dihydroxylated compounds by what must be two successive hydroxylation events: m-xylene was converted to 2,4-dimethylresorcinol and p-xylene was converted to 2,5-dimethylhydroquinone. The rigorous structural identification of 2,4-dimethylresorcinol and 2,5-dimethylhydroquinone demonstrates that DK17 can perform distinct regioselective hydroxylations depending on the position of the substituent groups on the aromatic ring.