3-Hydroxyphenylacetate 6-hydroxylase was purified 70-fold from a Flavobacterium sp. grown upon phenylacetic acid as its sole carbon and energy source. The presence of FAD and dithiothreitol during purification is essential for high recovery of active enzyme. SDS/PAGE of purified enzyme reveals a single band with a minimum molecular mass of 63 kDa. Analytical gel-filtration, sedimentation-equilibrium and sedimentation-velocity experiments indicate that the purified enzyme exists in solution mainly as a dimer, containing 1 molecule non-covalently bound FAD/subunit. 3-Hydroxyphenylacetate 6-hydroxylase utilizes NADH and NADPH as external electron donors with similar efficiency. The enzyme shows a narrow substrate specificity. Only the primary substrate 3-hydroxyphenylacetate is hydroxylated efficiently, yielding 2,5-dihydroxyphenylacetate as a product. During turnover, the substrate analogues 3,4-dihydroxyphenylacetate and 4-hydroxyphenylacetate are partially hydroxylated, exclusively at the 6' (2') position. The physiological product 2,5-dihydroxyphenylacetate acts as an effector, strongly stimulating NAD(P)H oxidation. The activity of 3-hydroxyphenylacetate 6-hydroxylase is severely inhibited by chloride ions, competitive to the aromatic substrate. In the native state of enzyme, two sulfhydryl groups are accessible to 5,5'-dithiobis(2-nitrobenzoate). Titration with stoichiometric amounts of either 5,5'-dithiobis(2-nitrobenzoate) or mercurial reagents completely blocks enzyme activity. Inactivation by cysteine reagents is inhibited by the substrate 3-hydroxyphenylacetate. The original activity is fully restored by treatment of the modified enzyme with dithiothreitol. The N-terminal amino acid sequence of the enzyme lacks the consensus sequence GXGXXG, found at the N-termini of all flavin-dependent external monooxygenases sequenced so far. The amino acid composition of 3-hydroxyphenylacetate 6-hydroxylase is also presented.