Sarcoplasmic reticulum (SR) was isolated from rabbit skeletal muscle as a microsomal fraction, and kinetic properties of its ATPase reaction were studied. The following results were obtained. 1. SR showed a remarkable extra-ATP splitting in the presence of 3mM oxalate and 0.1 mM Ca++. In the absence of oxalate or in the presence of both oxalate and minute amounts of Triton, the ATPase was elevated by Ca++, and the rate, which was equal to that of extrasplitting in the presence of oxalate and Ca++, was maintained for a long time. It was concluded that ATPase is activated by Ca++ outside of SR membrane. 2. The ATPase activity decreased with increase in the concentration of EGTA to 0.1 mM, above which it remained at a constant level. This ATPase was called the basal ATPase, and the Ca++-dependent ATPase was defined as the difference between the total ATPase and the basal ATPase activity. 3.The Lineweaver-Burk plot of Ca++-dependent ATPase consisted of two lines. In the presence of 50μM Ca++, the apparent Michaelis constant over high concentrations of ATP was 50μM, while the apparent constant over low concentrations of ATP was 0.7 μM. 4.The dependency on Ca++ concentration (0—1μM) of ATPasew activity at 25 μM ATP was insensitive to the Mg++ concentration from 0.5 to 5mM. This result shows that the Mg.ATP complex is “true”true substrate of the Ca++-dependent ATPase. Kinetic studies of Ca++-dependent ATPase at low concentrations of ATP showed that the rate (v) at low concentrations of Ca++ is given by where V, Φ1 and Φ2 are constants independent of the concentrations of Ca++ and ATP. The values of Φ1 and Φ2 were about 5 μM and 0.4 μM,respectively. The value of V fluctuated considerably from one preparation to the other, and its average value was about 0.2 μMoles P1 per mg. SR protein per minute. At high concentrations of Ca, ATPase was inhibited by Ca**, and v was found to be given by 5. When SR was incubated with γ-32pP labelled ATP, it incorporated P within 5 seconds after the addition of ATP. The protein-phosphate complex was TCA-stable. The amount decreased gradually to zero with splitting of ATP. The incorporation was dependent on Ca** and was inhibited by NEM. The 32P-incorporation in the presence of Ca** and ATP was decreased rapidly when Ca** was removed by the addition of EGTA. The 32P-incorporation increased with increasing Ca-concentration even in the range of saturation for Ca**-dependent ATPase. The amount of 32P incorporated at sufficiently high concentrations of Ca4 was about 6 moles per l07g. of SR protein. 6. The following reaction scheme was proposed to explain the properties of Ca**-dependent ATPase mentioned in 4 and 5; where EP1 Ca and EP2 are two phosphorylated intermediates and only the former is TCA-stable. ATP is the Mg.ATP complex, and ECa2 is an inactive complex of enzyme with Ca** 7. Kinetic studies of Ca**-dependent ATPase at high concentrations of ATP showed that the rate (v) is given by when the concentration of Ca** is low. The values of Φ1 and Φ21 were different from those at low concentrations of ATP, and were 160 μM and 0.13 μM, respectively. The value of V was in the order of 0.5 μMoles per mg. SR protein per minute. 8. In spite of great difference in the values of Φ1 and V, the dependencies of Ca**-dependent ATPase at high concentrations of ATP on pH, temperature and NEM were very similar to those at low concentrations of ATP. These results suggest that the reaction mechanism of ATPase itself in the presence of a high concentration of ATP is the same as that in the presence of a low concentration of ATP, but the binding strength of ATP and the maximum rate are affected by the addition of a large amount of ATP.