Isolated pancreatic islets from the rat incubated with 28 mM glucose have been found to secrete more insulin and to synthesize greater amounts of arachidonate lipoxygenase and cyclooxygenase products than islets incubated with 3 mM glucose. This effect was not apparent in studies examining metabolism of radiolabeled arachidonate and was revealed only when the metabolites were quantitated with mass spectrometric measurements. That the glucose-induced synthesis of arachidonate metabolites may participate in insulin secretion was suggested by studies with inhibitors of arachidonate metabolism. Eicosa 5,8,11,14 tetrynoic acid (ETYA) suppressed glucose-induced insulin secretion by 63-74% at a concentration (20 microM) which inhibited the synthesis of arachidonate lipoxygenase and cyclooxygenase products by 90%. Indomethacin (10 microM) completely prevented islet synthesis of cyclooxygenase products but did not influence glucose-induced insulin secretion. Although indomethacin did not inhibit the conversion of exogenous, 3H-labeled arachidonate to [3H]12-HETE, it did significantly inhibit (41-72%) the synthesis of 12-HETE from endogenous precursor. This is presumed to reflect indirect effects of indomethacin on hydrolysis of arachidonate from phospholipids, as recently reported in platelets. These studies constitute the first demonstration that glucose stimulates the synthesis of a lipoxygenase product (12-HETE) from endogenous arachidonate by isolated islets, and that suppression of 12-HETE synthesis with ETYA reduces glucose-induced insulin secretion from isolated islets.