Human serum plasma paraoxonase/arylesterase exhibits a genetic polymorphism for the hydrolysis of paraoxon. One allelic form of the enzyme hydrolyzes paraoxon slowly with a low turnover number and the other(s) hydrolyzes paraoxon rapidly with a high turnover number. Chlorpyrifos-oxon, the active metabolite of the insecticide chlorpyrifos (Dursban), is also hydrolyzed by plasma arylesterase/paraoxonase. A specific assay for measuring hydrolysis of this compound is described. This assay is not subject to interference by the esterase activity of serum albumin. The Km for chlorpyrifos-oxon hydrolysis was 75 microM. Hydrolysis was inhibited by phenyl acetate, EDTA, and organic solvents. Enzyme activity required calcium ions and was stimulated by sodium chloride. Hydrolysis was optimized by using methanol instead of acetone to dissolve substrate. Unlike the multimodal distribution of paraoxonase, the distribution of chlorpyrifos-oxonase activity failed to show clear multimodality. An improvement in the assay for hydrolysis of paraoxon by plasma arylesterase/paraoxonase was achieved by elimination of organic solvents. Plotting chlorpyrifos-oxonase activity vs paraoxonase activity for a human population using the new assay conditions provided an excellent resolution of low activity homozygotes from heterozygotes for this allele. A greater than 40-fold difference in rates of chlorpyrifosoxon hydrolysis observed between rat (low activity) and rabbit sera (high activity) correlated well with the reported large differences in LD50 values for chlorpyrifos in these two animals, consistent with an important role of serum paraoxonase in detoxification of organophosphorus pesticides in vivo.