In spontaneously diabetic GK rats, insulin secretion from pancreatic beta cells in response to glucose is selectively impaired, probably due to deficient intracellular metabolism of glucose and impaired closure of K_ATP channels during glucose stimulation. By using electrically permeabilized islets of GK rats, we explored the functional modulations in exocytotic steps distal to the rise in [Ca²⁺]i in the diabetic condition. At 30 nmol/l Ca²⁺ (basal conditions) insulin release was similar between GK and non-diabetic control Wistar rats. In response to 3.0 μmol/l Ca²⁺(maximum stimulatory conditions), insulin release was significantly augmented in permeabilized GK islets (p < 0.01). Raising glucose concentrations from 2.8 to 16.7 mmol/l further augmented insulin release induced by 3.0 μmol/l Ca²⁺ from permeabilized control islets (p < 0.001), but had no effect on that from permeabilized GK islets. The stimulatory effect of glucose on insulin release from permeabilized control islets was partly inhibited by 2,4-dinitrophenol, an inhibitor of mitochondrial oxidative phosphorylation (p < 0.01). The hyperresponse to Ca²⁺ in GK islets may play a physiologically compensatory role on the putative functional impairment both in [Ca²⁺]i rise and energy state in response to glucose in diabetic β cells, and may explain the relative preservation of insulin release induced by non-glucose depolarizing stimuli, such as arginine, from pancreatic islets in non-insulin-dependent diabetes mellitus.