The preincubation of the rat blood cell membranes in the presence of low Ca2+ levels causes an irreversible inhibition of the Ca2+-stimulated ATPase activity. The inactivation is dependent on the Ca2+ concentration and the apparent Ki is identical to the Ca2+ concentration needed to reach the half-maximal activity of the enzyme. This fact and the energy of activation (Ea = 13.8 K cal/mol) for the inhibition suggest that Ca2+ inactivates the Ca2+-stimulated ATPase by binding to the same site which it normally occupies to activate the enzyme. It is concluded that the Ca2+-stimulated ATPase is in a dynamic equilibrium between two states: a stable ATP-bound state and an unstable ATP-free state.