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 using Ca²⁺ imaging, electrophysiology and cell-column perifusion. Although caffeine inhibited K⁺ channels, this action appeared to be unrelated caffeine-induced GTH-II release, since the later was insensitive to TEA. The effects of caffeine were also not mediated by the cAMP/PKA pathway. Instead, caffeine-evoked GTH-II response was Ca²⁺ signal-dependent, as it was abolished by BAPTA loading. Accordingly, caffeine generated Ca²⁺ signals that began localized 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 SERCA 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.