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Chlorpyrifos (CPS) is a broad-spectrum organophosphate insecticide that is neurotoxic in humans. Chlorpyrifos oxon (CPO) is a toxic metabolite of CPS that is produced by CYP2B6. In this study, we examined the variability of CPS metabolism resulting from single-nucleotide polymorphisms in CYP2B6. Wild-type CYP2B6 (CYP2B6.1) and two variants each with a single amino acid substitution: CYP2B6.5 (R487C) and CYP2B6.8 (K139E) were co-expressed together with human NADPH-dependent cytochrome P450 reductase in Escherichia coli (E. coli). Both of the CYP2B6 variants were successfully expressed in E. coli. The conversion of CPS to CPO by the CYP2B6 variants was analyzed with high-performance liquid chromatography. K m and V max of the reaction by CYP2B6.1 were 18.50±2.94μM and 17.07±1.15mol/min/mol P450, respectively. The CYP2B6 variants produced CPO with the following kinetic parameters: K m for CYP2B6.5 and CYP2B6.8 were 20.44±6.43 and 44.69±9.97μM, respectively; and V max were 1.10±0.10 and 1.77±0.26mol/min/mol P450, respectively. These results indicate that the amino acid substitutions in the CYP2B6 variants suppressed the metabolic activation of CPS. CYP2B6 variants have altered capacity to bioactivate CPF and may affect individual susceptibility of CPF., (Copyright © 2017. Published by Elsevier Inc.)