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Mast cells are hematopoietic-derived immune cells that possess numerous cytoplasmic granules containing immune mediators such as cytokines and histamine. Antigen stimulation triggers mast cell granule exocytosis, releasing granule contents in a process known as degranulation. We have shown that Rho GTPase signaling is an essential component of granule exocytosis, however, the proteins that regulate Rho GTPases during this process are not well defined. Here we examined the role of Rho guanine-nucleotide dissociation inhibitors (RhoGDIs) in regulating Rho GTPase signaling using RBL-2H3 cells as a mast cell model. We found that RBL-2H3 cells express two RhoGDI isoforms which are primarily localized to the cytosol. Knockdown of RhoGDI1 and RhoGDI2 greatly reduced the levels of all Rho GTPases tested: RhoA, RhoG, Rac1, Rac2, and Cdc42. The reduction in Rho GTPase levels was accompanied by an increase in their membrane-localized fraction and an elevation in the levels of active Rho GTPases. All RhoGDI knockdown strains had altered resting cell morphology, although each strain was activation competent when stimulated. Live cell imaging revealed that the RhoGDI1/2 double knockdown (DKD) strain maintained its activated state for prolonged periods of time compared to the other strains. Only the RhoGDI1/2 DKD strain showed a significant increase in granule exocytosis. Conversely, RhoGDI overexpression in RBL-2H3 cells did not noticeably affect Rho GTPases or degranulation. Based on these results, RhoGDIs act as negative regulators of Rho GTPases during mast cell degranulation, and inhibit exocytosis by sequestering Rho GTPases in the cytosol. [ABSTRACT FROM AUTHOR]