Secretagogues of rat peritoneal mast cells, such as mastoparan and compound 48/80, induce mast cell exocytosis by activating directly the guanosine triphosphate-binding proteins that are required for exocytosis. The introduction of a synthetic peptide that corresponds to the carboxyl-terminal end sequence of Gα_i3 into the cells specifically blocked this secretion. Similar results were obtained when antibodies to this peptide were introduced. The Gα_i3 was located in both the Golgi and the plasma membrane, but only the latter source of Gα_i3 appeared to be essential for secretion. These results indicate that Gα_i3 functions to control regulated exocytosis in mast cells.