Aspirinated human platelets labeled with 32PO4 showed a 1.7-fold increase in [32P]IP3 when stimulated with ADP. ADP-stimulated mobilization of internal Ca2+ and phosphorylation of myosin were enhanced in the presence of extracellular Ca2+ but the increase in IP3 was not significantly affected by external Ca2+. The Ca2+ ionophore, ionomycin, elevated internal Ca2+ and induced myosin phosphorylation without a detectable change in IP3. These results indicate that the mechanism of ADP stimulation of human platelets is similar to that of other platelet agonists and supports the theory that IP3 functions to liberate internal Ca2+.