In rat pancreatic islets, a rise in D-glucose concentrations increases the oxidation of hexose-derived acetyl residues relative to glycolytic flux, an effect possibly attributable, in part at least, to the activation of key mitochondrial dehydrogenases by Ca2+ accumulated in the mitochondria of glucose-stimulated islet cells. The effects of non-nutrient insulinotropic agents upon D-[6-14C]glucose oxidation and D-[5-3H]glucose utilization were investigated. At an intermediate concentration of D-glucose (6 mM), the oxidation of D-[6-14C]glucose was unaffected by hypoglycemic sulfonylureas, an organic Ca2+ agonist, a cholinergic agent, forskolin, theophylline and cytochalasin B. At a higher concentration of the hexose (17 mM), however, the 14CO2/3H2O production rate was decreased by organic and inorganic Ca(2+)-antagonists and by ouabain, whilst being increased by NH+4 (10 mM) and aminooxyacetate. These findings suggest that the preferential stimulation of oxidative events in the Krebs cycle is largely independent of the rate of insulin release, and not merely consequential to the stimulation of Ca2+ inflow into the B-cell. It might be regulated, in a feedback process, by the rate of ATP utilization and, both directly and indirectly, by the mitochondrial redox state. The glucose-induced mitochondrial accumulation of Ca2+ and subsequent activation of the Krebs cycle appear to require an increase in both cytosolic Ca2+ activity and ATP availability.