In guinea pig antral mucous cells, acetylcholine (ACh) stimulates the Ca²⁺-regulated exocytosis, which has a characteristics feature: an initial transient phase followed by a sustained phase. The effects of guanosine 3',5'-cyclic monophosphate (cGMP) on ACh-stimulated exocytosis were studied in guinea pig antral mucous cells using video microscopy. Cyclic GMP enhanced the frequency of ACh-stimulated exocytotic events, while cGMP alone did not induce any exocytotic events under the ACh-unstimulated condition. Cyclic GMP did not stimulate either Ca²⁺ mobilization or cAMP accumulation. The Ca²⁺ dose-response studies demonstrated that cGMP shifted the dose-response curve upward with no shift to the lower-concentration. This indicates that cGMP increased maximal responsiveness of the Ca²⁺-regulated exocytosis, but not the Ca²⁺ sensitivity. Moreover, under a condition of ATP depletion by dinitrophenol (DNP) or anoxia (N₂ bubbling) ACh evoked only a sustained phase in exocytotic events with no initial transient phase. However, ACh evoked an initial transient phase followed by a sustained phase with addition of cGMP before ATP depletion, whereas only a sustained phase was evoked in a case of cGMP addition after ATP depletion. Thus, cGMP-induced enhancement in ACh-stimulated exocytotic events requires ATP, suggesting that cGMP modulates ATP-dependent priming of Ca²⁺-regulated exocytosis in antral mucous cells. In conclusion, cGMP increases the number of primed granules via acceleration of the ATP-dependent priming, which enhances the Ca²⁺-regulated exocytosis stimulated by ACh.