Okadaic acid (OA), a potent inhibitor of protein phosphatases type 1 and type 2A, inhibited thrombin-induced platelet aggregation (IC 5 0 = 0.8 μM), [ 1 4 C]serotonin release and increase in intracellular Ca 2 + ([Ca 2 + ] i ) in the same dose dependence. In the absence of thrombin OA increased the phosphorylation of 50-kDa protein and 20-kDa myosin light chain (MLC20). The 50-kDa protein phosphorylation was accomplished within a shorter time period and at a lower concentration than was the MLC20, OA decreased the thrombin-induced phosphorylation of 47-kDa protein and MLC20, although phosphorylation of MLC20 reincreased at higher concentrations of OA (5-10 μM). Since type 2A phosphatase is more sensitive to OA than type 1, these results suggest that type 2A phosphatases are involved in the regulation of Ca 2 + signaling in thrombin-induced platelet activation.