Acute exposure of pancreatic islets to interleukinl-1β results in an increase in insulin release, while an extension of the exposure time induces a functional suppression and eventually, destruction of the B-cells. We have recently suggested that the interleukin-1β induced inhibition of islet function is mediated through an impairment in oxidative metabolism. The aim of the current study was to investigate if the acute, stimulatory effects of interleukin-1β on islet function could also be related to changes in the substrate metabolism. For this purpose, rat islets were exposed for 90–120 min to 30 pmol/l human recombinant interleukin-1β (biological activity of 2.5 U/ml) and their function and metabolism characterized during this period. The cytokine did not increase insulin release in the presence of 1.7 or 5.5 mmol/l glucose but in both the presence of 16.7 mmol/l glucose or 10 mmol/l leucine + 2 mmol/l glutamine there was a 50% increase in insulin release. Interleukin-1β exposure increased the oxidation of D-[U-¹⁴C]glucose at 5.5 mmol/l glucose by 25% and at 16.7 mmol/l glucose by 60%. Carbohydrate and amino acid metabolism were further examined in the presence of D-[5-³H] glucose, D-[6-¹⁴C]glucose, [1-¹⁴C]pyruvate, L-[U-¹⁴C]glutamine, L-[U-¹⁴C]leucine and L-[1-¹⁴C]leucine. There was no difference between control islets and interleukin-1β exposed islets in terms of D-[5-³H]glucose utilization or [1-¹⁴C]pyruvate decarboxylation, but the oxidation of D-[6-¹⁴C]glucose was increased by 64% in the interleukin-1β exposed islets. There was also an interleukin-1β induced 45–60% increase in the decarboxylation of L-[1-¹⁴C]leucine and oxidation of L-[U-¹⁴C]leucine and L-[U-¹⁴C]glutamine, all intramitochrondrial events. The stimulation of insulin release by interleukin-1β in the presence of 16.7 mmol/l glucose was abolished in islets incubated in Ca²⁺ depleted medium, but the rate of D-[6-¹⁴C] glucose oxidation remained elevated (47% increase at 16.7 mmol/l glucose). These data indicate an increase in substrate metabolism at the mitochondrial level during acute exposure of rat pancreatic islets to interleukin-1β. The increase in oxidative events can explain the observed interleukin-1β induced increase in insulin release during glucose stimulation. Furthermore, these findings raise the possibility that mitochondria are primary targets of interleukin-1β action in the B-cells.