In the present study, we investigated the role of cAMP-dependent protein kinase in the process of Ca²⁺ uptake and release from platelet-derived membrane vesicles enriched in the dense tubular system. It was found that these membrane vesicles contain endogenous cAMP-dependent protein kinase and that stimulation of protein kinase by cAMP resulted in the phosphorylation of a single protein band (22 kDa). Addition of cAMP-dependent protein kinase produced effects on vesicle Ca²⁺ accumulation which were dependent on the Ca²⁺ concentration in the incubation medium. Specifically, at low extravesicular Ca²⁺ concentrations, cAMP-dependent protein kinase (10–100 μg/ml) produced a dose-dependent stimulation of Ca²⁺ uptake, however, a similar stimulation was not observed at high extravesicular Ca²⁺ concentrations. When endogenous protein kinase was blocked by the addition of protein kinase inhibitor, (2–160 nM) there was a dose-dependent inhibition of Ca²⁺ uptake at both low and high concentrations of extravesicular Ca²⁺. Furthermore, the addition of protein kinase inhibitor at steady state caused a rapid and dose-dependent release of vesicle-accumulated Ca²⁺. Studies on the phosphorylation profile of vesicle protein indicated that protein kinase inhibitor (80 and 160 nM) was capable of inhibiting the phosphorylation of the 22-kDa protein within 15 s. Finally, the ability of thromboxane A₂ to cause Ca²⁺ release was inhibited by the addition of cAMP-dependent protein kinase (1 mg/ml). These findings suggest that cAMP-dependent protein kinase is not only a major determinant in the accumulation of Ca²⁺ by the dense tubular system, but may play an important role in the process of intraplatelet Ca²⁺ release by physiologic agents such as thromboxane A₂.