In this paper we have used streptolysin O (SLO)-permeabilized human platelets to examine the G-protein(s) that control Ca2+-independent secretion from alpha and dense-core granules. As shown for electropermeabilized platelets, Ca2+ alone stimulated a concentration-dependent increase in 5-hydroxytryptamine (5-HT) (dense-core-granule marker) and platelet-derived growth factor (PDGF) (alpha-granule marker) release from the SLO-permeabilized cells. The EC50 values of Ca2+-dependent 5-HT and PDGF release were 5 microM and 10 microM respectively. Guanosine 5'-[gamma-thio]triphosphate (GTP[S]) (100 microM) stimulated Ca2+-independent release from both alpha and dense-core granules. In contrast, AlF4- had no effect on Ca2+-independent release from either alpha or dense-core granules. Neither GTP[S] nor AlF4- appeared to have a significant effect on Ca2+-dependent release from alpha and dense-core granules. GTP[S] can activate both heterotrimeric and low-molecular-mass G-proteins, whereas AlF4- activates only heterotrimeric G-proteins. Our results, therefore suggest that secretion in the human platelet is regulated by a small G-protein. Both GTP[S]- and Ca2+-dependent secretion were effected by extending the time between permeabilization with SLO and stimulation of secretion. GTP[S]-stimulated secretion from alpha and dense-core granules decreased rapidly after permeabilization. In contrast, Ca2+-dependent 5-HT and PDGF release ran down at a much lower rate. These observations indicate that GTP[S] and Ca2+ act through parallel pathways to stimulate secretion from SLO-permeabilized platelets.