Metabolism of arachidonic acid (AA) via the cyclooxygenase pathway reduces glucose-stimulated insulin release. However, metabolism of AA by the lipoxygenase pathway and the consequent effects on insulin secretion have not been simultaneously assessed in the endocrine islet. Both dispersed endocrine cell-enriched pancreatic cells of the neonatal rat, as well as intact islets of the adult rat, metabolized [(3)H]AA not only to cyclooxygenase products (prostaglandins E(2), F(2alpha), and prostacyclin) but also to the lipoxygenase product 12-hydroxyeicosatetraenoic acid (12-HETE). 12-HETE was identified by coelution with authentic tritiated or unlabeled 12-HETE using four high performance liquid chromatographic systems under eight mobile-phase conditions and its identity was confirmed by gas chromatography/mass spectrometry using selected ion monitoring. The predominant effect of exogenous AA (5 mug/ml) was to stimulate insulin release from pancreatic cells grown in monolayer. This effect was concentration- and time-dependent, and reversible. The effect of AA upon insulin release was potentiated by a cyclooxygenase inhibitor (indomethacin) and was prevented by either of two lipoxygenase inhibitors (5,8,11,14-eicosatetraynoic acid [ETYA] and BW755c). In addition, glucose, as well as two structurally dissimilar agents (the calcium ionophore A23187 and bradykinin), which activate phospholipase(s) and thereby release endogenous AA in several cell systems, also stimulated insulin secretion. The effects of glucose, glucagon, bradykinin and high concentrations of A23187 (5 mug/ml) to augment insulin release were blocked or considerably reduced by lipoxygenase inhibitors. However, a lower concentration of the ionophore (0.25 mug/ml), which did not appear to activate phospholipase, was resistant to blockade. Exogenous 12-HETE (up to 2,000 ng/ml) did not alter glucose-induced insulin release. However, the labile intermediate 12-hydroperoxy-ETE increased insulin release. Furthermore, diethylmaleate (which binds intracellular glutathione and thereby impedes conversion of the lipoxygenase intermediates hydroperoxy-ETE and leukotriene A(4) to HETE and leukotriene C(4), respectively) potentiated the effect of glucose and of exogenous AA. Finally, 5,6-epoxy, 8,11,14-eicosatrienoic acid (a relatively stable epoxide analogue of leukotriene A(4)) as well as two other epoxy-analogues, potentiated glucose-induced insulin release. We conclude that dual pathways of AA metabolism exist in islet endocrine cells and have opposing regulatory effects on the beta cell-an inhibitory cyclooxygenase cascade and a stimulatory lipoxygenase cascade. Labile products of the latter pathway may play a pivotal role in stimulus-secretion coupling in the islet.