The bacterial phosphotriesterase (PTE) isolated from Flavobacterium sp. can catalyze the cleavage of the P-O bond in a variety of organophosphate triesters and has been shown to be, an effective catalyst for the degradation of toxic organophosphate esters. Ethyl 4-nitrophenyl phenylphosphonothioate (EPN) is a chiral organophosphate. Optical isomers of EPN show differences in their toxicity. R-EPN is known to be more toxic to hens and houseflies than S-EPN. We determined the K-i value of each enantiomer toward electric eel acetylcholinesterase. R-EPN (K-i = 6 mu M) inhibited acetylcholinesterase much more effectively than S-EPN (K-i = 52 mu M) did in vitro. Since PTE has been found to hydrolyze only the S-isomer of EPN, we attempted to alter the enantioselectivity of PTE in order to degrade toxic EPN enantiomer effectively. When PTE hydrolyzed EPN in the presence of dimethyl sulfoxide (DMSO), enzymatic activity toward S-EPN decreased linearly, but enzymatic activity toward R-EPN increased as a function of DMSO concentration. At 20% DMSO, the maximum activity was observed. The kinetic parameters of PTE to EPN isomers clearly indicated that in the presence of 20% DMSO, the enantioselectivity of PTE changed. The K-m value for R-EPN decreased from 0.24 to 0.03 mM, and the V-max value increased from 0.25 to 0.60 U/mg of protein. V-max/K-m values indicated that PTE preferred R-EPN over S-EPN in the presence of DMSO by a factor of 2.