This report describes the effects of gonadotropin-releasing hormone (GnRH; gonadoliberin) and an agonist, [D-Ala6, des-Gly10]GnRH ethyl amide (GnRHa), on phospholipid metabolism in rat granulosa cells isolated from mature Graafian follicles. As indicated by the incorporation of 32PO4, GnRHa rapidly (less than 2 min) stimulated the labeling of phosphatidic acid and phosphatidylinositol but had no effect on the labeling of other phospholipids. Increased phosphatidylinositol labeling was also observed when myo-[2-3H]inositol was incubated with granulosa cells in the presence of GnRHa. Increases in labeling were dependent on the dose of GnRH and time of incubation. Thyrotropin-releasing hormone and a specific GnRH antagonist had no effect on labeling, but a GnRH antagonist prevented the stimulatory action of GnRH. In addition, treatment with GnRHa slightly increased the levels of phosphatidylinositol (15%) in 60-min incubations but had no effect on the levels of other phospholipids. Significant increases in progesterone accumulation were observed after 30 min of incubation with GnRHa, and further increases were correlated with the time of incubation. The stimulatory action of GnRH on phospholipid metabolism and progesterone accumulation was not related to increases in cyclic nucleotide accumulation. In incubations lasting up to 30 min, GnRHa had no effect on cAMP accumulation. However, a transient decrease in cGMP levels was observed in response to GnRHa. These studies suggest that the rapid and specific effects of GnRH on phospholipid metabolism in rat granulosa cells represent early events in the action of GnRH.