An o-xylene-utilizing Rhodococcus, strain B3, was isolated from enrichments with o-xylene. The pathway for o-xylene degradation was investigated by simultaneous adaptation experiments, studies of product formation by a mutant and fortuitous oxidation studies using trimethylbenzene isomers as substrates. Two pathways were found to operate simultaneously and both were inducible. The first pathway involved the oxidation of a methyl group to form 2-methyl benzyl alcohol, followed by oxidation via the corresponding acid to 3-methylcatechol. The second pathway involved oxidation of the aromatic ring to form a dimethylcatechol. The bulk of the evidence suggests that the initial reaction was catalysed by a monooxygenase rather than a dioxygenase, and that 2,3-dimethylphenol was produced as an intermediate.