We have examined the effects of 2,3,5-trimethyl-6-(12-hydroxy-5,10-dodecadiynyl)-1,4-benzoquinone (AA861), a selective inhibitor of 5-lipoxygenase, on the action of cholecystokinin (CCK) and other secretagogues in the stimulation of amylase secretion from dispersed rat pancreatic acini. AA861 inhibited amylase secretion caused by CCK, carbamylcholine (carbachol), bombesin or calcium ionophore A23187 but failed to affect amylase secretion by vasoactive intestinal peptide or 12-O-tetradecanoyl-phorbol 13-acetate. Inhibition by AA861 of CCK or carbachol-induced amylase secretion was confined to the relatively lower concentrations of these secretagogues. AA861 did not inhibit receptor binding of CCK or alter the cellular calcium mobilization induced by CCK. In kinetic studies, AA861 was effective only on amylase secretion from pancreatic acini incubated with CCK for more than 5 min. Indomethacin, a known inhibitor of cyclooxygenase, did not affect the amylase secretion caused by all secretagogues used. These results indicate that the 5-lipoxygenase pathway of arachidonate metabolism may be involved in the actions of calcium-dependent secretagogues of amylase secretion in rat dispersed pancreatic acini, especially for sustaining stimulation of amylase secretion by CCK.