Prostaglandin I2 (PGI2) and sodium nitroprusside (SNP) induce a rapid decay of the thrombin-promoted increase of [Ca2+]i in aspirin-treated platelets incubated in the absence of external Ca2+. The mechanism of their effect was studied with a new method which utilizes ionomycin to increase [Ca2+]i, followed by bovine serum albumin (BSA) to remove the Ca2+ ionophore. The rapid decay of [Ca2+]i after BSA is mostly due to the reuptake into the stores, since it is strongly inhibited by the endomembrane Ca2+-ATPase inhibitor thapsigargin. PGI2 and SNP are without effect on the BSA-promoted decay both with and without thapsigargin, showing that they do not affect the activity of the Ca2+-ATPases. The fast decay of [Ca2+]i after BSA is decreased by thrombin which produces the Ca2+ releaser inositol 1,4,5-trisphosphate (InsP3), thus counteracting the activity of the endomembrane Ca2+ pump. When added after thrombin, PGI2 and SNP accelerate the BSA-activated decay of [Ca2+]i. However, under the same conditions, they do not decrease the concentration of InsP3. In saponin-permeabilized platelets, cAMP and cGMP counteract the Ca2+ release induced by exogenous InsP3. Their inhibitory effect disappears at high InsP3 concentrations. This demonstrates that PGI2 and SNP potentiate Ca2+ reuptake by inhibiting the InsP3 receptor. Two bands of approximately 260 kDa are recognized by a monoclonal antibody recognizing the C-terminal region of the InsP3 receptor. Both are phosphorylated rapidly, the heavier more intensely, in the presence of PGI2 and SNP. The phosphorylation of the InsP3 receptor is fast enough to be compatible with its involvement in the inhibition of the receptor by cyclic nucleotides.