The preparation and properties of a Mg 2+-dependent, (Na + + K +)-stimulated ATPase from platelets are described. The enzym system require Mg 2+ for activity, is stimulated maximally by the presence of Na + and K + in a ratio of 11.5/1, has a pH optimum 7.30–7.40, and is half-maximally inhibited by ouabain at a concentration of 0.15 μM. The K m for ATP is approx. 0.4 mM. Enzyme activity is localized in both the “membrane” and “membrane-granule” subcellular fractions. Mg 2+-dependent, (Na + + K +)-stimulated ATPase activity is inhibited by ADP (which induces platelet aggregation) and by P i. The inhibition by ADP is apparently not competitive with respect to ATP. Mg 2+-dependent, K +-stimulated p- nitrophenyl phosphatase activity (a model for the externally-oriented K +-phosphatase portion of Mg 2+-dependent, (Na + + K +)-stimulated ATPase) is also inhibited by ADP. GDP, which does not induce aggregation of platelets, has no effect on the Mg 2+-dependent, (Na + + K +)-stimulated ATPase activity. It is postulated that ADP interacts with the external platelet membrane surface to inhibit Mg 2+-dependent, (Na + + K +)-stimulated ATPase activity with a resultant decrease in cation transport and membrane potential. This phenomenon may play a role in platelet aggregation.