In antral mucous cells, acetylcholine (ACh, 1µM) activates Ca2+-regulated exocytosis, consisting of an initial peak that declines rapidly (initial transient phase) followed by a second slower decline (late phase) lasting during ACh stimulation. We have reported that the initial phase is enhanced by PPARα in antral mucous cells (1). However, at present, it still remains uncertain how PPARα enhances CaCa2+-regulated exocytosis in antral mucous cells. The aim of this study is to clarify the mechanism following the PPARα activation in antral mucous cells. Guinea pigs were anesthetized by pentobarbital-Na (70 mg/kg, ip). Antral mucous cells were isolated by a collagenase digestion. The exocytotic events were observed by video-microscopy. The procedures and protocols for these experiments were performed in accordance with the Guidelines of the Animal Research Committee of Osaka Medical College and the Guiding Principles for the Care and Use of Animals in the Field of Physiological Sciences (Physiological Society of Japan). GW7647 (a PPARα agonist) enhanced the frequency of initial phase stimulated by 1 µM ACh. In the presence of GW6471 (a PPARα blocker), GW7647 did not enhance the initial phase, and rather the frequency of the initial phase was reduced to about 65 % of that seen in the absence of GW7647. However, GW6471 produced a delayed, but transient, increase in the frequency of late phase. A PKG inhibitor has been reported to produce a similar delayed, but transient, increase in the late phase, since the inhibition of PKG suppresses PDE2 leading to cAMP accumulation by inhibiting cAMP degradation. We examined the effects of a PKG inhibitor on the CaCa2+-regulated exocytosis enhanced by GW7647. In the presence of a PKG inhibitor or a NOS inhibitor, GW7647 did not enhance the initial phase, and rather the frequency of the initial phase was reduced to about 65% of that seen in the absence of GW7647. However, a PKG inhibitor or a NOS inhibitor produced a delayed, but transient, increase in the frequency of the late phase. In the presence of GW6471, 8BrcGMP or NOC12 (an NO donor) still enhanced the Ca2+-regulated exocytosis without any delayed increase in the frequency of the late phase. This suggests that PPARα stimulates NO synthesis leading to cGMP accumulation. Moreover, 1 µM ACh or GW7647 increased NO production and cGMP contents in the antral mucosae. Analyses of Western blotting and immunohistochemistry demonstrated that NOS1 exists in antral mucous cells. Thus, PPARα stimulates NOS1 leading to NO synthesis and then NO accumulates cGMP resulting in the enhancement of Ca2+-regulated exocytosis in antral mucous cells. A novel autocrine mechanism mediated via PPARα maintains CaCa2+-regulated exocytosis.