Krebs II ascites cells were incubated with [3H] or [14C] choline in the presence or in the absence of Clostridium welchii phospholipase C (PLC). At enzyme concentrations where cell lysis remained limited, PLC specifically enhanced phosphatidylcholine (PC) biosynthesis, as shown by comparison with [14C] ethanolamine. Further analysis revealed that the stimulating effect of PLC remained limited to 1,2-diacyl-sn-glycero-3-phosphocholine (diacyl-GPC) and 1-alkenyl-2-acyl-GPC, whereas the biosynthesis of 1-alkyl-2-acyl-GPC, the putative precursor of platelet activating factor (PAF-acether) remained unchanged. These differences reflect different localizations of the three PC subclasses in the plasma membrane and are discussed in relation to the regulation mechanism of PC biosynthesis.