Stimulation of immunoglobulin E (IgE)-sensitized mast cells by multivalent antigen triggers a cascade of intracellular signaling events that results in granule exocytosis as a principal outcome. Granule exocytosis results in the release of histamine and other inflammatory mediators of the allergic response, and this process is mediated by Ca2+mobilization and activation of protein kinase C (PKC). MARCKS, a major PKC substrate, has been implicated in granule exocytosis. MARCKS has a polybasic effector domain (ED) that associates strongly with phosphatidylinositol 4,5bisphosphate (PIP 2 ) and other phosphoinositides at the inner leaflet of the plasma membrane. Using real-time fluorescence imaging, we observed antigen-stimulated oscillatory recruitment of EGFP-tagged PKC[beta][IOTA] to the plasma membrane in a process that is synchronous with the oscillatory displacement of mRFP-MARCKS-ED. To investigate the role of PKC-mediated MARCKS phosphorylation in granule exocytosis, we created a MARCKS-ED mutant that cannot be phosphorylated. We observed that MARCKS-EDSA4 delays the onset of Ca2+ mobilization that is dependent on PIP 2 hydrolysis to produce IP 3 in response to antigen, but it does not inhibit store-operated Ca2+ entry (SOCE) activated by the SERCA pump inhibitor thapsigargin. Under these conditions, we found that granule exocytosis stimulated by antigen as well as by thapsigargin is substantially inhibited. Our results provide strong evidence that phosphoinositides participate in the terminal steps of granule exocytosis, and they support the hypothesis that PKC normally regulates access of PIP 2 to multiple proteins in both Ca2+ mobilization and granule exocytosis. We also investigated the role of endogenous MARCKS and found that its knockdown resulted in reduced Ca2+ and degranulation responses to antigen, suggesting a positive regulatory role for endogenous MARCKS in these processes. These results contrast with the inhibition by MARCKS-ED SA4 in these processes, and they suggest that full length, endogenous MARCKS may be regulating multiple pathways. We hypothesize that endogenous MARCKS positively regulates antigenstimulated store-operated Ca2+ entry and granule exocytosis due to its association with Ca2+ bound calmodulin under stimulated conditions. These findings provide useful mechanistic insights into the roles of PKC, MARCKS and PIP 2 in the intricate regulation of Ca2+ mobilization and granule exocytosis in stimulated mast cell signaling.