The non-hydrolyzable GTP analogue, guanosine 5'-O-(3-thiotriphosphate) (GTPγS) and cyclic AMP potentiated the Ca 2 + -evoked secretion of α-melanocyte-stimulating hormone (α-MSH) from permeabilized neurointermediate lobe (IL) cells of rat pituitary gland. The enhancement by Mg-GTPγS (100 μM) and cyclic AMP (1 μM) depended on the intracellular CA 2 + concentration (EC 5 0 = 4.8 +/- 1.8 and 4.6 +/- 1.7 μM; mean +/- SE, with and without Mg-GTPγS and cyclic AMP, respectively). A similar effect was observed with guanine nucleotide triphosphate (GTP and GppNHp). Mg was absolutely required for this event. Neither Mg-GTPγS nor cyclic AMP alone was effective in potentiating α-MSH secretion. GDPβS blocked the Mg-GTPγS (100 μM) and cyclic AMP augmented secretion of α-MSH. Neither neomycin (which affects the process of inositol 1,4,5-triphosphate-mediated Ca 2 + mobilization) or colchicine (which influences microtubule assembly) had an effect on the cyclic AMP and Mg-GTPγS potentiation of α-MSH secretion. These data suggest that the GTP-binding protein may be involved in the regulation of α-MSH secretion after Ca 2 + entry into the cells, since the intracellular environment is controlled in the permeabilized cells.