Acetylcholine-evoked secretion from the parotid gland is substantially potentiated by cAMP-raising agonists. A potential locus for the action of cAMP is the intracellular signaling pathway resulting in elevated cytosolic calcium levels ([Ca2+]i). This hypothesis was tested in mouse parotid acinar cells. Forskolin dramatically potentiated the carbachol (CCh)-evoked increase in [Ca2+]i, converted oscillatory [Ca2+]i changes into a sustained [Ca2+]i increase, and caused subthreshold concentrations of CCh to increase [Ca2+]i measurably. This potentiation was found to be independent of Ca2+ entry and inositol 1,4,5-trisphosphate (InsP3) production, suggesting that cAMP-mediated effects on Ca2+ release was the major underlying mechanism. Consistent with this hypothesis, dibutyryl-cAMP dramatically potentiated InsP3-evoked Ca2+ release from streptolysin-O-permeabilised (SL-O) cells. Furthermore, type II InsP3 receptors (InsP3R) were shown to be directly phosphorylated by a protein kinase-A (PKA)-mediated mechanism after treatment with forskolin. In contrast, no evidence was obtained to support direct PKA-mediated activation of ryanodine receptors (RyRs). However, inhibition of RyRs in intact cells, demonstrated a role for RyRs in propagating Ca2+ oscillations and amplifying potentiated Ca2+ release from InsP3Rs. These data indicate that potentiation of Ca2+ release is primarily the result of PKA-mediated phosphorylation of InsP3Rs, and may largely explain the synergistic relationship between cAMP-raising agonists and ACh-evoked secretion in the parotid. In addition, this report supports the emerging consensus that phosphorylation at the level of the Ca2+ release machinery is a broadly important mechanism by which cells can regulate Ca2+-mediated processes.