The present study was undertaken to determine if acute stress induced by exposure to ether resulted in the presence of β-cell-tropic factors in rat plasma and if this insulinotropic activity was increased by pertussis toxin. Rats pretreated with pertussis toxin (5 μg/kg, 5 days previously) showed marked hyperinsulinemia, but only after exposure to ether before blood sampling. This hyperinsulinemia was not modified by adrenal demedullation. Effects on insulin secretion were assessed by incubation of plasma (diluted with Krebs buffer) with collagenase-isolated rat pancreatic islets. When blood was collected by decapitation from normal rats, the subsequently prepared plasma (12.5% to 50%) profoundly inhibited insulin release from rat isolated islets. This inhibition was probably mediated by catecholamines, since it was not seen with plasma from adrenal-demedullated rats and was prevented by α2-adrenoceptor-blocking drugs. Plasma from adrenal-demedullated, pertussis toxin-treated rats stimulated insulin secretion (by 60%) when the donor rats had been exposed to ether before blood sampling. It is suggested that stress may result in the presence of circulating β-cell-tropic factors, which may contribute to the acute stress-induced hyperinsulinemia seen in pertussis toxin-treated animals.