The A23187 calcium ionophore strongly inhibits the hydrosmotic response to vasopressin. On the contrary neither exogenous cAMP nor theophylline-induced hydrosmotic response are affected by Ca++-ionophore. The effects obtained with A23187 suggest that calcium ions modulate vasopressin-induced osmotic water flow by interfering with a pre-cAMP step. The increase in intracellular calcium concentration induced by A23187, in fact, may inhibit the rate of cAMP formation by interfering with the vasopressin-stimulated adenylate cyclase system. This inhibition seems to be restricted to a locus proximal to the catalytic subunit of adenylate cyclase, whereas the hydrosmotic response to forskolin, a non-hormonal activator, is not affected by calcium ionophore addition. In order to clarify whether calcium ions act directly on the adenylate cyclase system or activate a more complex pathway, we studied the interaction between calcium and prostaglandins in modulating the hydrosmotic response to vasopressin. Direct measurements by radioimmunoassay of PGE2 release in the serosal medium show that A23187 notably increases PGE2 release. Furthermore, the inhibition of prostaglandin biosynthesis by hydrocortisone (a phospholipase inhibitor) or by indomethacin and naproxen (agents that inhibit arachidonic acid oxygenase) results in augmented vasopressin-stimulated water flow and prevents the inhibitory effect of the ionophore. Collectively these results strongly suggest that the effects obtained with A23187 on hydrosmotic response to ADH, are closely linked to calcium-stimulated PGE2 biosynthesis.