The active metabolites (oxons) of phosphorothionate insecticides can be detoxified via Aesterase hydrolysis. Two enzymes with A-esterase activity have been isolated from rat serum. Whole serum was applied to anion exchange gel (DEAE Sepharose Fast Flow) and incubated (1 hr). Tris-HCl buffer (0.05 M; pH 7.7, at 5 °) containing 0.25 M NaCl was added to the slurry and incubated. The decant, containing low A-esterase activity but a high protein concentration, was discarded. Further displacement of A-esterase from DEAE gel was achieved with 1.0 M NaCl in 0.05 M Tris-HCl buffer (pH 7.7 at 5 °). Following desalting and concentration, further separation was achieved by gel filtration (Sephacryl S-100 HR) and two sequential preparative scale isoelectric focusings. Final fractions contained two proteins of high molecular mass (one about 200 kDa and one between 137 and 200 kDa). The apparent range of isoelectric points for the two enzymes was 4.5 to 5.6. Following native-PAGE analysis, activity stains with β-naphthyl acetate and Fast Garnet GBC in the presence of paraoxon (10 −5 M) verified that A-esterase activity was associated with both proteins. Spectrophotometric assay detected A-esterase activity toward paraoxon, chlorpyrifos-oxon, and phenyl acetate in the final preparation.