Homogenates of isolated pancreatic islets contain 40-70 times as much flavin-linked glycerol-3-phosphate dehydrogenase (EC 1.1.99.5) as homogenates of whole pancreas, liver, heart, or skeletal muscle when the activity is assayed with either iodonitrotetrazolium or with dichloroindophenol as an electron acceptor. Intact mitochondria from islets release 3HOH from [2-3H]glycerol phosphate 7 times faster than do skeletal muscle mitochondria. The activity of the cytosolic, NAD-linked, glycerol phosphate dehydrogenase (EC 1.1.1.8) in pancreatic islets is comparable to that of the mitochondrial dehydrogenase so a glycerol phosphate shuttle is possible in pancreatic islets. Diazoxide, an inhibitor of insulin release in vivo and in vitro, inhibits the islet mitochondrial glycerol phosphate dehydrogenase in all three of the assays mentioned above at concentrations that inhibit insulin release and CO2 formation from glucose by isolated pancreatic islets. Diazoxide does not inhibit the dehydrogenase in mitochondria from skeletal muscle, liver, and heart. A slight inhibition in mitochondria from whole pancreas can be accounted for as inhibition of the islet dehydrogenase because no inhibition is observed in mitochondria from pancreas of rats treated with alloxan, an agent that causes diabetes by destroying pancreatic beta cells. The results of this study are compatible with the hypothesis that the mitochondrial glycerol phosphate dehydrogenase has a key role in stimulus-secretion coupling in the pancreatic beta cell during glucose-induced insulin release.