4,4'-Dithiocyanatostilbene-2,2'-disulfonic acid (DIDS), an inhibitor of the volume-sensitive anion channel, was used to investigate the role of this channel in the stimulation of rat pancreatic beta-cells by glucose and by tolbutamide. Glucose-stimulated electrical activity in beta-cells was markedly and reversibly inhibited by DIDS. The increase in cytosolic [Ca2+] and stimulated insulin release evoked by glucose were also inhibited by DIDS. In contrast to its inhibitory effect on glucose-induced beta-cell activity, DIDS had no effect on electrical activity, the rise in [Ca2+]i or insulin release induced by tolbutamide. DIDS failed to increase beta-cell input conductance, an index of whole-cell K(ATP) channel activity, or the rate of efflux of 86Rb+ from perifused islets, a measure of net K+ permeability. Furthermore, DIDS had no effect on intracellular pH or on regulatory volume increase following exposure of cells to hypertonic solutions, indicating that the effects of DIDS were not the result of inhibition of Cl- transport systems. It is suggested that the DIDS-induced repolarization is caused by inactivation of the volume-sensitive anion channel. The stimulation of beta-cell electrical and secretory activity by glucose, but not tolbutamide, may therefore involve activation of the anion channel.