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 G"alpha"i3 into the cells specifically blocked this secretion. Similar results were obtained when antibodies to this peptide were introduced. The G"alpha"i3 was located in both the Golgi and the plasma membrane, but only the latter source of G"alpha"i3 appeared to be essential for secretion. These results indicate that G"alpha"i3 functions to control regulated exocytosis in mast cells.