We identified an aldoxime dehydratase (Oxd) gene in the 5'-flanking region of the nitrile hydratase-amidase gene cluster in the photoreactive iron-type nitrile hydratase-producer, Rhodococcus sp. N-771. The enzyme showed 96.3%, 77.6%, and 30.4% identities with the Oxds of Rhodococcus globerulus A-4, Pseudomonas chlororaphis 1323, and Bacillus sp. OxB-1, respectively. The enzyme was expressed in Escherichia coli under the control of the lac- or T7 promoters in its intact and His(6)-tagged forms, purified, and characterized. The enzyme had heme b as a prosthetic group, catalyzed a stoichiometric dehydration of aldoxime into nitrile, and exhibited the highest activity at neutral pH and at around 30degreesC similar to the known Oxd from Bacillus sp. OxB-1. The activity was enhanced by reducing agents, such as Na2S, Na2S2O4, 2-mercaptoethanol, and L-cysteine and supplementary additions of electron acceptors such as flavins, sulfite ion, and vitamin K-3. The effect of various chemicals on the enzyme activity was different in the presence and absence of the reducing reagent, Na2S. The enzyme preferentially acts on aliphatic-type substrates and the substrate specificity of the enzyme coincides with that reported for nitrile hydratase produced by the strain.