The role of calcium in triggering prostaglandin and thromboxane synthesis was studied in several systems with ionophores of different ion specificities. Divalent cationophore A23187 stimulates prostaglandin and thromboxane production by washed human platelets in a concentration-dependent manner (0.3-9 μM). A23187 also induces an antimycin A-insensitive burst in oxygen utilization which is partially blocked by 5 mM aspirin or 10 μM indomethacin. Under our conditions, A23187 (up to 10 μM) does not appear to damage platelet membranes since it does not cause appreciable loss of lactate dehydrogenase or β-glucuronidase. Mono- and divalent cationophore X537A also stimulates platelet thromboxane B2 production and oxygen utilization, but monovalent cationophores nigericin, monensin A, A204, and valinomycin have no effect. The synthesis of prostaglandins E2, D2, and F2α by rat renal medulla mince is stimulated by 1 and 5 μM A23187 without changes in tissue ATP content, lactate output, or K+ efflux. X537A, monensin A, and nigericin (all 5 μM) stimulate both prostaglandin output and K+ efflux from renal medulla, while 5 μM valinomycin or A204 has no effect on either. None of the ionophores stimulates renomedullary prostaglandin production if calcium is omitted from the incubation medium. A23187 also stimulates prostaglandin production by human lymphoma cells, rat stomach and trachea preparations, and guinea pig polymorphonuclear leukocytes. These observations suggest a major role for Ca2+ in stimulating prostaglandin and thromboxane biosynthesis, and also indicate that prostaglandin and/or thromboxane release may partially mediate some of the previously described effects of ionophores on cells and tissues.