Summary 1. The activation of mitochondrial adenosine triphosphatase (EC 3.6.1.4) by Ca2+ was studied using membranes from deoxycholate-disrupted rat-liver mitochondria as the source of enzyme. With. 3 mM ATP and 1–12 mM Ca2+, pH-activity curves in the range of pH 7.4 to 10.0 showed a broad optimum between 8.0 and 9.0. 2. With 3 mM ATP, maximum ATPase activity shifted from a Ca2+ to ATP ratio of approx. 4:1 to a ratio of approx. 2:1 as the pH was increased from 7.0 to 10.0. 3. When ATPase activity was determined between pH 7.0 and 10.0, plots of 1/vversus 1/[ATP] resulted in a series of straight lines with values for Km which decreased as the pH was raised to 9.5. Values for vmax increased as the pH was raised to 8.5 and then decreased as the pH was raised to 10.0. 4. The kinetics of ATPase activation by Ca2+ at pH 8.5 indicate that CaATP2-is the “true” or “active” substrate for the enzyme, and that free ATP and free Ca2+ react with the enzyme. Free Ca2+ ions appear to increase the rate of breakdown of the ATPase-CaATP complex. Free Ca2+ ions also decrease the affinity of the enzyme for CaATP. Free ATP appears to inhibit breakdown of the ATPase-CaATP complex and also to act as a competitive inhibitor. 5. The results are discussed in relation to the similarities and differences of mitochondrial ATPase activation by Ca2+ and by Mg2+. A comparison of the action of the two ions indicates that the affinity of both CaATP and free Ca2+ for the enzyme is significantly greater than that of MgATP and free Mg2+. However the affinity of free ATP for the enzyme appears to be considerably less than when Ca2+ is the activating cation.