The thiophospholipid 1,2-dipalmitoyl-sn-glycero-3-thiophosphocholine (DPPsC) was shown to be a mixture of two diastereomers by 31P nuclear magnetic resonance. The isomer that resonates at the lower field in CDCl3 (56.12 ppm) was designated as isomer A and the other (resonates at 56.07 ppm) as isomer B. Phospholipase A2 from four different sources (bee venom, Naja naja venom, Crotalus adamanteus venom, and porcine pancreas) was shown to hydrolyze the isomer B of DPPsC specifically, whereas phospholipase C from two different sources (Bacillus cereus and Clostridium perfringens) hydrolyzes isomer A specifically. So that the two diastereomers could be separated, DPPsC(A + B) was first digested with phospholipase A2 to give 1-palmitoyl-sn-glycero-3-thiophosphocholine (MPPsC) (which is designated as isomer B of MPPsC) and the unreacted DPPsC(A). Reacylation of MPPsC(B) gave pure DPPsC(B). The properties of DPPsC(A) and DPPsC(B) were investigated by 31P, 13C, 1H, and 14N nuclear magnetic resonance (NMR). 1H and 13C NMR showed that both isomers in methanol solution have conformational properties similar to those of the natural phospholipid, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine. On the other hand, the two isomers A and B showed small but significant differences in the chemical shifts of the carbon in the chiral carbon center and the phosphorus in the chiral phosphorus center.