Erythrocyte membranes prepared by three different procedures showed (Mg2+ + Ca2+)-ATPase activities differing in specific activity and in affinity for Ca2+. The (Mg2+ + Ca2+)-ATPase activity of the three preparations was stimulated t o different extents by a Ca2+-dependent protein activator isolated from hemolysates. The Ca2+ affinity of the two most active preparations was decreased as the ATP concentration in the assay medium was increased. Lowering the ATP concentration from 2 mM t o 2-200 pM or lowering the Mg:ATP ratio t o less than one shifted the (Mg2+ + Ca2+)-ATPase activity in stepwise hemolysis membranes from mixed "high" and "low" affinity t o a single high Ca2+ affinity. Membranes from which soluble proteins were extracted by EDTA (0.1 mM) in low ionic strength, or membranes prepared by the EDTA (1-10 mM) procedure, did not undergo the shift in the Ca2+ affinity with changes in ATP and MgCl2 concentrations. The EDTA-wash membranes were only weakly activated by the protein activator. It is suggested that the differences in properties of the (Mg2+ + Ca2+)-ATPase prepared by these three procedures reflect differences determined in part by the degree of association of the membrane with a soluble protein activator and changes in the state of the enzyme to a less activatable form. Key words: (Mg2 + + Ca2 +)-ATPase, erythrocyte membranes, endogenous protein activator Controversy exists as t o the properties, number and function of (Mg2+ + Ca2+)-ATPase(s) present in erythrocyte membranes. Quist and Roufogalis [ 1 , 2 ] , employing a stepwise hemolysis procedure for preparation of erythrocyte membranes, identified mixed "high" and "low" Ca2+ affinity (Mg2+ + Ca2+)-ATPase activity. A correlation of ATPase