Abstract 1. 1. The subcellular localization of Ca 2+/Mg 2+-stimulated ATPase in human platelets was studied. The effect of thrombin on the enzyme was also investigated. 2. 2. Characterization of the ATPase activity in platelet lysates revealed some similarities with actomyosin ATPase in its response to divalent cations. 3. 3. Both Ca 2+- and Mg 2+-stimulated ATPases were found to be localized in a granular fraction containing the platelet-bound adenine nucleotides. 4. 4. Thrombin did not cause the release of Ca 2+/Mg 2+-activated ATPase from washed human platelets. 5. 5. A slight inhibition of Ca 2+-stimulated ATPase occurred 10 sec after exposure of washed human platelets to thrombin. 6. 6. That Ca 2+/Mg 2+-stimulated ATPase occurs in granules containing adenine nucleotides seems established. However, although a slight drop in ATPase activity occurred after the addition of thrombin to washed platelets, no evidence of ATPase activation during the release phenomenon was obtained.