The hydrolytic activities of organophosphorus hydrolase (OPH) toward nerve agents were investigated by using mutant OPH enzymes constructed by site-directed mutagenesis of the gene. The amino acids at position 136, 254, and 257 are known to influence the enzyme activity. The nucleotide sequencing of the OPH gene cloned in our previous study revealed that the deduced amino acid at position 254 was tyrosine (Tyr) instead of histidine (His) that was reported to reside at this position by other research groups. In the present study, such key amino acids were substituted to construct five mutant enzymes, and their hydrolytic activities were compared with that of the wild type enzyme. The activity assays proved that the substitution of Tyr at 254 to His led to the remarkable enhancement of the activities toward paraoxon and also toward VX, which could be decomposed by the wild type OPH only slightly. This mutant enzyme hydrolyzed most of the nerve agents almost completely, and approximately half of the VX during 20 min when the enzyme was activated with 10 mM CoCl2. The cobalt ion could activate the mutant OPH more efficiently than zinc ion, but the difference of the activation ability was not significant when other mutant OPH enzymes were activated with these divalent metal ions. The present results suggest that a practical decontamination system for nerve agents could be established using the mutant OPH with enhanced activity.