Recent studies have revealed that SNARE proteins are involved in exocytotic granular content release in mast cells as well as in neurotransmitter release in neural cells. However, the proteins that regulate the structure and activity of SNARE proteins in mast cells are not well understood. Munc18 is one such regulatory protein that plays a crucial role in neurotransmitter release. In this study, we investigated the role of Munc18 and its mechanism for regulating exocytotic release (degranulation) in rat basophilic leukemia cells (RBL-2H3). We obtained RBL-2H3 cells that express a low level of Munc18-2 and found that degranulation was remarkably inhibited in knockdown cells without any change in the expression level of syntaxins or Ca(2+) mobilization. We also observed the behavior of secretory granules in a single cell, and found no significant changes in their number and distribution in Munc18-2 knockdown cells. Using chimera proteins fused with fluorescent proteins, we demonstrated that Munc18-2 interacted with syntaxin-3, but not with syntaxin-4, in vivo. Interestingly, this interaction occurred not only on plasma membrane but also on secretory granules, suggesting that Munc18-2 may regulate granule-granule fusion as well as granule-plasma membrane fusion. These observations suggest that Munc18-2 together with syntaxin-3 regulate degranulation positively during the process of membrane fusion between secretory granules and plasma membrane, rather than during processes that regulate the number or behavior of secretory granules.