Introducing non-hydrolysable analogues of GTP into the cytosolic compartment of mast cells results in exocytotic secretion through the activation of GTP binding proteins. The identity and mechanism of action of these proteins are not established. We have investigated the effects of Rho GDP dissociation inhibitor (RhoGDI) on exocytosis induced by guanosine 5'-O-(3-thiotriphosphate) (GTP-gamma-S) in rat mast cells, introducing the protein into cells by means of a patch pipette and recording the progress of exocytosis by monitoring cell capacitance. To allow time for the protein to enter the cells and find its correct location, stimulation was provided 5-10 min after patch rupture by photolysing caged GTP-gamma-S included in the pipette solution. When bovine RhoGDI was introduced into mast cells, exocytosis was inhibited at concentrations of 200-400 nM for native protein and 800 nM to 8 microM for the recombinant form. Protein denatured by heat or N-ethylmaleimide treatment did not inhibit. In permeabilized cells, recombinant RhoGDI increased the rate at which cells lose their ability to respond to GTP-gamma-S. These data demonstrate that one or more small GTP binding proteins of the Rho family has a central role in the exocytotic mechanism in mast cells.