In order to examine the role of phospholipids in the activation of membrane bound Ca2+/Mg2+ ATPase, the activities of Ca2+ ATPase and Mg2+ ATPase were studied in heart sarcolemma after treatments with phospholipases A, C and D. The Mg2+ ATPase activity was decreased upon treating the sarcolemmal membranes with phospholipases, A, C and D; phospholipase A produced the most dramatic effect. The reduction in Mg2+ ATPase activity by each phospholipase treatment was associated with a decrease in the Vmax value without any changes in the Ka value. The depression of Mg2+ ATPase in the phospholipase treated preparations was not found to be due to release of fatty acids in the medium and was not restored upon reconstitution of these membranes by the addition of synthetic phospholipids such as lecithin, lysolecithin or phosphatidic acid. In contrast to the Mg2+ ATPase, the sarcolemmal Ca2+ ATPase was affected only slightly by phospholipase treatments. The greater sensitivity of Mg2+ ATPase to phospholipase treatments was also apparent when deoxycholate-treated preparations were employed. These results indicate that glycerophospholipids are required for the sarcolemmal Mg2+ ATPase activity to a greater extent in comparison to that for the Ca2+ ATPase activity and the phospholipids associated with Mg2+ ATPase are predominantly exposed at the outer surface of the membrane.