Platelet thromboxane A2 (TXA2) synthesis is an important pathway of platelet reactivity. We report that in thrombin-stimulated platelets, PP1/PP2A serine/threonine phosphatases regulate phospholipase A2 (cPLA2) activity, which is required for TXA2 synthesis. Two mechanisms are involved: (a) constitutively active PP1/PP2A regulate cPLA2 phosphorylation, and (b) PP1/PP2A activity mediates agonist-induced increase in cytosolic Ca2+ ([Ca2+]i). Inhibition of PP1/PP2A with okadaic acid (OA) induces cPLA2 phosphorylation but reduces Ca2+ responses: release from intracellular stores and influx through the plasma membrane, particularly that mediated by store-mediated Ca2+ entry (SMCE). A significant correlation (r = 0.64) exists between OA-regulated [Ca2+]i and TXA2 synthesis. Okadaic acid-induced decrease in SMCE and the associated TXA2 synthesis are mediated by a reduction in protein-tyrosine phosphorylation. This reduction is not due to inhibition of tyrosine kinases but rather to an OA-mediated increase in tyrosine phosphatases. This is the first study to report that PP1/PP2A phosphatases are involved in the regulation of the two key elements in eicosanoid synthesis, [Ca2+]i and cPLA2 phosphorylation. Moreover, PP1/PP2A regulation of [Ca2+]i and tyrosine phosphorylation may be important for other calcium-dependent processes and/or signal transduction mechanisms in platelets.