Even in the initial phase of the reaction, 45Ca2+ taken up by the sarcoplasmic reticulum (SR)** was not released on addition of a large amount of EGTA, and was not exchanged with non-radioactive Ca2+. About 2 moles of Ca2+ were taken up by the SR per mole of Ca**-dependent hydrolysis of ATP under conditions where the release of Ca2+ from the SR was insignificant; i.e. in the initial phase of the reaction in the presence of high or low concentrations of ATP or during all phases of the reaction in the presence of low concentrations of ATP. All the hydrogen ion released during Ca2+-uptake could be attributed simply to hydrolysis of ATP. Phosphate derived from ATP did not pass through the membrane.