Gonadotropin-releasing hormone (GnRH) stimulates luteinizing hormone (LH) release and cyclic guanosine 3’,5-cyclic monophosphate (cGMP) production in rat anterior pituitary cells through a calcium-dependent activation mechanism that involves increased phospholipid turnover and liberation of arachidonic acid. In enriched pituitary gonadotrophs, LH release was stimulated by arachidonic acid and its oxygenated metabolite, 5-hydroxy-6,8, l 1,14-eicosatetraenoic acid (5-HETE), in a dose-dependent manner. The prominent LH responses of purified gonadotrophs to arachidonic acid suggest that the secretory actions of arachidonate are exerted primarily on the gonadotroph and do not involve the participation of other pituitary cell types. Preincubation of pituitary cells with stimulatory concentrations of arachidonic acid for up to 120 min did not alter the subsequent LH responses elicited by GnRH, indicating that the secretory mechanism was unimpaired by arachidonate treatment and that no cross-desensitization occurs during sequential exposure of gonadotrophs to the two stimuli of LH release. Cyclic adenosine 3’,5-monophosphate (cAMP) production was stimulated by 10 µ M arachidonic acid to the same degree (about 2-fold) as by GnRH, but did not parallel the progressive LH response to higher arachidonate concentrations. cGMP production was initially stimulated by addition of arachidonic acid but returned to the control value after 5 min, whereas GnRH typically elicited a prolonged cGMP response. In contrast to the calcium-independent action of arachidonic acid, the stimulatory effect of 5-HETE on LH release required the presence of extracellular Ca<sup>2+</sup> , as previously observed for GnRH. These findings demonstrate that arachidonic acid and its metabolite, 5-HETE, partially reproduce the actions of GnRH upon LH release and cyclic nucleotide production. The several similarities between the effects of GnRH and those of arachidonic acid and 5-HETE suggest that arachidonic acid and/or its lipoxygenase metabolites participate in the mechanism of LH secretion.