We have attempted to delineate the effect of glyburide on the regulation of cytosolic-free calcium concentrations, [Ca2+]i, in normal rat pancreatic islet cells. In the presence of extracellular calcium (1 mmol/L), glyburide increased [Ca2+]i from 70 nmol/L to 260 nmol/L in a dose-dependent manner. The maximal effect was seen at a concentration of 2 mumol/L with half-maximal stimulation observed at .25 mumol/L. The effect of glyburide (.25 mumol/L) was inhibited 90% by the calcium channel blocker, verapamil (30 mumol/L). At a maximally effective concentration of glyburide (2 mumol/L), the inhibitory effect of verapamil was only 17%. In the absence of extracellular calcium, glyburide increased [Ca2+]i from 55 nmol/L to 107 nmol/L, indicating its ability to mobilize intracellular calcium stores. These results correlated well with the ability of glyburide (2 mumol/L) to stimulate insulin secretion both in the presence (from 38 +/- 5 mumol/L/10 islets to 131 +/- 28 microU/10 islets) and in the absence (from 49 +/- 4 microU/10 islets to 93 +/- 7 microU/10 islets) of extracellular Ca2+. The present observations suggest that glyburide promotes calcium influx via voltage-dependent calcium channels and may mobilize intracellular calcium stores.