The gram-positive Rhodococcus erythropolis strain S1 was found to utilize o-, m-, and p-hydroxyphenylacetic acids as sole carbon sources. Each isomer of monohydroxyphenylacetate was degraded via the homogentisate pathway, which contained a reduced glutathione independent-isomerase. Three monohydroxyphenylacetate monooxygenases, o-hydroxyphenylacetate 5-hydroxylase, m-hydroxyphenylacetate 6-hydroxylase, and p-hydroxyphenylacetate 1-hydroxylase, were purified to homogeneity from strain S1. Each enzyme was a 45-kDa monomeric NADH-dependent monooxygenase containing FAD, and all three appeared to belong to the p-hydroxybenzoate hydroxylase-class as regards the flavin-containing aromatic compound monooxygenase family. However, the three enzymes differed greatly in terms of substrate specificity.