Two organophosphorus compound hydrolases constitutively expressed in Arthrobacter sp. strain B-5 that was capable of degrading various organophosphorus insecticides were purified from the cells’ cytosol. The enzymes were similarly composed of a single subunit with a molecular weight of 45,000 Da, but had different isoelectric points of 3.6 and 3.9. In addition, the hydrolases were found to possess identical N-terminal amino acid sequences, which showed 51.6% amino acid identity with the N-terminus of the aryldialkylphosphatase from Nocardia sp. strain B-1 and revealed no significant homology for any other proteins. The pH and temperature optima for the activity of the enzyme mixture were 9.5 and 45 degrees C, respectively. The B-5 hydrolases were remarkably activated by ammonium ions and thiols such as 2-mercaptoethanol and dithiothreitol, and significantly inactivated by divalent cations such as CuCl2. Of all organophosphorus compounds tested, only isofenphos was rapidly hydrolyzed by the enzyme mixture, which cleaved an aryl phosphoestser bond in isofenphos and produced it hydrolysates, O-ethyl isopropyl phosphoramidothioate and isopropyl salicylate. The K-m and V-max values for isofenphos were 1.8 mu M and 9.4 U/mg protein, respectively. Since the other substrates possessing the phosphoramide bond such as butamifos, amiprophos-methyl, and acephate were not hydrolyzed very much by the enzymes, the bond would not play an important role on the hydrolytic activity. Thus, the high activity for isofenphos and a little hydrolysis of other substrates revealed that the enzymes from Arthrobacter sp. strain B-5 are novel type of organophosphorus compound hydrolase on substrate specificity.