The effects of cAMP accumulation evoked by acetylcholine (ACh) stimulation were studied in rat submandibular acinar cells by observing the exocytotic events, swelling of intercellular canaliculi (IC) and intracellular Ca2+ concentration ([Ca2+]i), which were monitored using an optical microscope. ACh stimulation evoked transient increases followed by sustained increases in the frequency of exocytotic events and IC swelling, while isoproterenol (isoprenaline; IPR) stimulation evoked sustained increases in these parameters. BAPTA treatment reduced the frequency of exocytotic events evoked by 5 µM ACh in the absence of extracellular Ca2+, and further addition of Rp-cAMPS or H-89 (protein kinase A (PKA) inhibitors) eliminated the remaining ACh-evoked responses (50 %). Addition of PKA inhibitors in the presence of extracellular Ca2+ reduced the frequency of exocytotic events evoked by 500 µM ACh in non-BAPTA-loaded cells. However, IC swelling evoked by 5 µM ACh was not affected by addition of PKA inhibitors, and was eliminated in BAPTA-loaded cells perfused with Ca2+-free solution. These results indicate that the IC swelling is regulated by [Ca2+]i and the frequency of exocytotic events is regulated by both [Ca2+]i and [cAMP]i during ACh stimulation. Addition of H-89 inhibited the capacitative Ca2+ entry into ACh-stimulated acinar cells. Biochemical analysis revealed that ACh stimulation increased the cAMP content in perfused submandibular glands. These results indicate that ACh stimulates the accumulation of cAMP in submandibular acinar cells and that this accumulation of cAMP modulates Ca2+-regulated exocytosis.