Abstract In the presence of MgCl 2 and ATP, the specific viscosity of suspensions of unsealed freezethawed erythrocyte membranes decreased slowly with time at 37 °C. The decrease in viscosity was found to be an index of Mg-ATP-specific induced folding of these membranes. Mg-ATP-dependent shape or viscosity changes were found to be highly temperature dependent and the viscosity of these membranes did not decrease in the presence of 2 m m 5′-adenyl imidodiphosphate and MgCl 2 . Cyclic AMP, NaCl, or KCl did not have any effect on the rate of Mg-ATP-induced viscosity decreases. The Mg-ATP-dependent viscosity decreases were inhibited 100% by 1 m m chlorpromazine or 1 m m N -ethylmaleimide. Mg-ATP-dependent viscosity decreases were half-maximally inhibited by 1 μ m Ca 2+ and completely inhibited by 3–5 μ m Ca 2+ . Ca 2+ (5 μ m ) also inhibited Mg 2+ -dependent phosphorylation 25 to 30% in these membranes. However, if these membranes were preincubated in the absence of Ca 2+ for greater than 10 min at 37 °C, 5 μ m Ca 2+ no longer inhibited Mg-ATP-dependent viscosity decreases and only inhibited Mg 2+ -dependent phosphorylation 5% in these preincubated membranes. Preincubation of these membranes at 37 °C for 10 min in the absence of Ca 2+ also resulted in the loss of approximately 40 to 50% of the high-Ca 2+ affinity Ca + Mg-ATPase activity. The presence of 5 μ m Ca 2+ in the preincubation medium protected against the loss of the inhibitory effect of Ca 2+ on Mg 2+ -dependent phosphorylation and Mg-ATP-dependent viscosity decreases. The presence of Ca 2+ in the preincubation medium also protected against the loss of Ca + Mg-ATPase activity in these membranes. It is hypothesized that freeze-thawed erythrocyte membranes contain a Ca 2+ phosphatase activity which is temperature labile in the absence of Ca 2+ and that this Ca 2+ phosphatase activity may be involved in the regulation of shape of these membranes. Also discussed is the possible relationship of this Ca 2+ phosphatase with Ca + Mg-ATPase activity and the problems inherent in studying Ca 2+ -regulated functions in freeze-thawed erythrocyte membranes.