The effects of the diacylglycerol lipase inhibitor 1,6-bis-(cyclohexyloximinocarbonyl-amino)-hexane (RHC 80267) and the phospholipase A 2 inhibitor N-(p-amylcinnamoyl)anthranilic acid (ACA) on insulin secretion and 86Rb + efflux in mouse pancreatic islets were studied. RHC 80267 (35 μM) and ACA (100 μM) inhibited glucose (16.7 mM)-induced insulin secretion, but did not inhibit insulin secretion induced by K + (40 mM) or the phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA; 0.16 μM). K + (40 mM) or TPA (0.16 μM) potentiated glucose (16.7 mM)-induced insulin secretion, and prevented inhibition of glucose (16.7 mM)-induced insulin secretion by RHC 80267 and ACA. In comparison, potentiation of glucose-induced insulin secretion by albumin-bound arachidonic acid (AA; 200 μM total; 10 μM free unbound) failed to counteract inhibition of glucose-induced insulin secretion by RHC 80267 or ACA, suggesting that inhibition of insulin secretion by these agents was not mediated by a decrease in AA accumulation in islets. Determination of 86Rb + efflux, a marker of K + channel activity, revealed that both RHC 80267 and ACA stimulated K + efflux from islets. These effects of RHC 80267 and ACA were observed at both 3.3 and 16.7 mM glucose and persisted in Ca 2+-free medium, suggesting that they may represent an opening of ATP-sensitive K + channels. RHC 80267-mediated stimulation of 86Rb + efflux was not mimicked by the diacylglycerol analog TPA (0.16 μM) and was not prevented by the diacylglycerol kinase inhibitor R 59022 (50 μM), suggesting that stimulation of 86Rb + efflux did not reflect a conditional increase in diacylglycerol or in phosphatidic acid upon inhibition of diacylglycerol lipase. In contrast, TPA (0.16 μM) attenuated RHC 80267 and ACA stimulation of 86Rb + efflux. Addition of AA (200 μM total; 10 μM free unbound) stimulated 86Rb + efflux, suggesting that stimulation of 86Rb + efflux by RHC 80267 and ACA was not due to a decrease in AA accumulation. This stimulation by AA was not dependent on AA metabolism because it persisted in the presence of the lipoxygenase inhibitor nordihydroguaiaretic acid (NDGA; 50 μM) or the cyclooxygenase inhibitor indomethacin (50 μM). In contrast to RHC 80267 and ACA, AA stimulation of 86Rb + efflux was attenuated in Ca 2+-free medium, probably implicating Ca 2+-sensitive K + channels in AA regulation of 86Rb + efflux. Parallel experiments with diazoxide (100 μM) revealed that RHC 80267 and ACA mimicked the effects of diazoxide, a specific activator of ATP-sensitive K + channels in islets, on both insulin secretion and 86Rb + efflux. In conclusion, it is suggested that RHC 80267 and ACA, independently of their action on AA release, may inhibit glucose-induced insulin secretion by the opening of ATP-sensitive K + channels in islets.