1. 1. The role of phospholipid in Ca 2+-stimulated ATPase activity of sarcoplasmic reticulum vesicles was reinvestigated using phospholipases A or C to degrade phospholipid. The byproducts of phospholipase A digestion were removed with a wash solution containing bovine serum albumin. Removal of 80–90 % of the phospholipid using phospholipase A led to loss of ATPase activity but had relatively little effect on the steady state level of phosphoenzyme. The formation of the phosphoenzyme, an intermediate in ATPase activity, was not blocked by the phospholipase A treatment, whereas the degradation of the intermediate was. Similar results were obtained with vesicles treated with phospholipase C. 2. 2. The requirement for phospholipid in the Ca 2+-stimulated ATPase was clearly demonstrated. Both addition of phospholipid to the assay and rebinding of phospholipid restored ATPase activity of vesicles depleted of lipid by phospholipase A. 3. 3. Ca 2+ is required for phosphoenzyme formation in both normal and lipid-depleted sarcoplasmic reticulum vesicles.