Recent reports of a pertussis-toxin (Ptx)-sensitive inhibition of glucose-induced insulin release by prostaglandin E2 (PGE2) in transformed beta-cells prompted us to look for the presence of prostaglandin-regulatable GTP-binding proteins (G-proteins) on the secretory granules of normal pancreatic islets. PGE2 (but not PGF2 alpha, PGA2, PGB2 or PGD2) stimulated in a concentration-dependent manner a high-affinity GTPase activity in the secretory-granule-enriched fractions of both normal rat and human islets. Similar results were found after sucrose-density-gradient-centrifugation-based isolation of secretory granules to those after a differential-centrifugation procedure. Half-maximal stimulation occurred at 800 nM PGE2, a concentration known to inhibit both phases of glucose-induced insulin secretion from pure beta-cell lines. The GTPase stimulatory effect of PGE2 was blocked virtually totally by Ptx pretreatment; it was not due to an effect on substrate binding since no measurable effect of PGE2 on binding of guanosine 5′-[gamma-[35S]thio]triphosphate was observed in cognate fractions. Other Ptx-sensitive inhibitors of insulin secretion (such as adrenaline or clonidine) also stimulated GTPase activity, suggesting that one (or more) inhibitory exocytotic G-proteins (i.e. a putative GEi) is located on the secretory granules. These studies demonstrate, for the first time in an endocrine gland, the presence of a regulatable G-protein, strategically located on the secretory granules where it might regulate the exocytotic cascade distal to both plasma-membrane events and the generation of soluble mediators of insulin secretion.