Cholinesterases (EC 3.1.1.8, acylcholine acylhydrolase) from the sera of man, dog and pig were purified 400–600-fold using a combination of ion-exchange and affinity chromatography. In a first approach, phosphonylation by soman was studied by using the half-resolved epimers C(+) P(±)-soman and C(−) P(±)-soman. The degradation of soman at the nanomolar level was followed in time by determining the remaining soman by capillary gas chromatography with NP detection. In the three sera investigated the P-(−)-epimer phosphonylates at a higher rate than its corresponding P(+)-counterpart and the stereoselectivity is greater for the C(+)-epimers than for the C(−)-epimers. Individual soman isomers were isolated from C(+)- and C(−)-epimers and quantified by gas chromatography. Second-order rate constants were determined for the phosphonylation of purified cholinesterase by isolated soman isomers. The C(+) P(−)-isomer has the highest phosphonylation rate for the three species; the other toxic isomer, C(−) P(−), has a five to ten-fold lower rate. The overall stereoselectivity is more marked in human cholinesterase than in canine. Porcine serum cholinesterase is phosphonylated by the P(−)-isomers at a slightly higher rate than the human enzyme.