The involvement of Ca2+ in the impaired insulin release of diabetic C57BL/KsJ-db/db mice was studied. Twenty-week-old severely hyperglycemic mice were compared to nondiabetic C57BL/KsJ mice as controls. Collagenase-isolated islets were maintained for 46 h in tissue culture allowing for equilibration at the same glucose concentration (8.3) mM). The insulin content of both types of islets was similar. In control islets preloaded during culture with 45Ca2+ glucose-induced insulin release was associated with increased 45Ca2+ effux. Islets from diabetic mice showed markedly reduced insulin response to glucose and a smaller increase in 45Ca2+ efflux. Because insulin release was strikingly potentiated by 3-isobutyl-1-methylxanthine (IBMX), even more than in control islets, there was no generalized release defect. In both types of islets, IBMX potentiation was accompanied by a further enhanced 45Ca2+ efflux, possibly suggesting that cAMP effects are associated with increased cytosol Ca2+% concentrations. As Ca2+ uptake was stimulated by glucose in both types of islets, a defect may lie in the mechanism by which glucose uses cellulr calcium to raise cytosol Ca2+ in the beta-cell of these diabetic mice.