Quinaldine 4-oxidase from Arthrobacter sp. Rü61a, an inducible molybdenum-containing hydroxylase, was purified to homogeneity by an optimized five-step procedure. Molecular oxygen is proposed as physiological electron acceptor. Electrons are also transferred to artificial electron acceptors with E'o > -8 mV. The molybdo-iron/sulfur flavoprotein regiospecifically attacks its N-heterocyclic substrates: isoquinoline and phthalazine are hydroxylated adjacent to the N-heteroatom at Cl, whereas quinaldine, quinoline, cinnoline and quinazoline are hydroxylated at C4. Additionally, the aromatic aldehydes benzaldehyde, salicylaldehyde, vanillin and cinnamaldehyde are oxidized to the corresponding carboxylic acids, whereas short-chain aliphatic aldehydes are not. Quinaldine 4-oxidase is compared to the two molybdenum-containing hydroxylases quinoline 2-oxidoreductase from Pseudomonas putida 86 [Tshisuaka, B., Kappl, R., Hüttermann, J. & Lingens, F. (1993) Biochemistry 32, 12928-12934] and isoquinoline 1-oxidoreductase from Pseudomonas diminuta 7 [Lehmann, M., Tshisuaka, B., Fetzner, S., Röger, P. & Lingens, F. (1994) J. Biol. Chem. 269, 11254-11260] with respect to the substrates converted and the electron-acceptor specificities. These dehydrogenases hydroxylate their N-heterocyclic substrates exclusively adjacent to the heteroatom. Whereas the aldehydes tested are scarcely oxidized by quinoline 2-oxidoreductase, isoquinoline 1-oxidoreductase catalyzes the oxidation of the aromatic aldehydes, although being progressively inhibited. Neither quinoline 2-oxidoreductase nor isoquinoline 1-oxidoreductase transfer electrons to oxygen. Otherwise, the spectrum of electron acceptors used by quinoline 2-oxidoreductase and quinaldine 4-oxidase is identical. However, isoquinoline 1-oxidoreductase differs in its electron-acceptor specificity. Quinaldine 4-oxidase is unusual in its substrate and electron-acceptor specificity. This enzyme is able to function as oxidase or dehydrogenase, it oxidizes aldehydes, and it catalyzes the nucleophilic attack of N-containing heterocyclic compounds at two varying positions depending on the substrate.