Modulation of Ca²⁺ stores with 10 mM caffeine stimulates robust secretion of gonadotropin (GTH-II) from goldfish gonadotropes. Although both endogenous forms of gonadotropin-releasing hormone (GnRH) utilize a common intracellular Ca²⁺ store, sGnRH, but not cGnRH-II, uses an additional caffeine-sensitive mechanism. We examined caffeine signaling by using Ca²⁺ imaging, electrophysiology, and cell-column perifusion. Although caffeine inhibited K⁺ channels, this action appeared to be unrelated to caffeine-induced GTH-II release, because the latter was insensitive to tetraethylammonium. The effects of caffeine also were not mediated by the cAMP/protein kinase A pathway. Instead, caffeine-evoked GTH-II responses were Ca²⁺ signal dependent because they were abolished by 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid loading. Caffeine generated localized Ca²⁺ signals that began near secretory granules. Surprisingly, caffeine-stimulated GTH-II release was insensitive to 100 µM ryanodine and, unlike GnRH action, was unaffected by inhibitors of voltage-gated Ca²⁺ channels or sarco(endo)plasmic reticulum Ca²⁺-ATPases. Collectively, these data indicate that caffeine-stimulated GTH-II release is not mediated by typical agonist-sensitive Ca²⁺ stores found in endoplasmic reticulum.