Pig gastric microsomal vesicles enriched in gastric H+,K+-ATPase and K+-pNPPase were digested with bee venom phospholipase A2 at 21 or 37 degrees C. The unattacked phospholipids were then related to the remaining enzyme activities, followed by reconstitution with microsomal phospholipids and the endogenous activator protein. Gastric K+-stimulated ATPase was nearly abolished within 10 min of phospholipase A2 treatment. A substantial amount of pNPPase activity remained unaffected under identical conditions. About 80% of the microsomal phosphatidylethanolamine was attacked by phospholipase A2 at both temperatures while 60 and 79% of the phosphatidylcholine was hydrolyzed at 21 and 37 degrees C, respectively. Analysis of the phospholipids revealed that phospholipase A2 attacked only the phosphatidylcholine and phosphatidylethanolamine molecules enriched in polyunsaturated fatty acids. Microsomal H+,K+-ATPase system inactivated by phospholipase A2 at 21 degrees C could be largely restored by the endogenous activator alone. On the other hand, those inactivated at 37 degrees C needed pretreatment with phosphatidylcholine before assaying with the activator protein for maximal reconstitution; phosphatidylethanolamine was totally ineffective in restoration of the enzyme activity. Analysis of the fatty acid composition of the lysophosphatidylcholine following phospholipase A2 treatment at 21 and 37 degrees C suggested involvement of some phosphatidylcholine molecules relatively enriched in saturated fatty acids and extremely poor in polyunsaturated fatty acids in gastric ATPase function. The data not only pointed out the importance of phosphatidylcholine and the endogenous activator in gastric microsomal H+,K+-ATPase reaction but also demonstrated considerable heterogeneity within the same species of microsomal phospholipids.