The regulation of growth hormone (GH) secretion by intracellular Ca²⁺ stores was studied in dissociated goldfish somatotropes. We characterized a caffeine-activated intracellular store that had been shown to mediate GH release in response to gonadotropin-releasing hormone. The peak response of caffeine stimulation was reduced by ~28% by 100 µM ryanodine in a use-dependent manner suggesting that the first 10 min of GH release is partially mediated by a caffeine-activated ryanodine receptor. The temporal sensitivities of caffeine- and dopamine-evoked GH release to blockade of Cd²⁺-sensitive Ca²⁺ channels were compared. We demonstrated that the initial phase of dopamine-evoked release was dependent on Ca²⁺ channels, whereas the initial phase of caffeine-evoked release was sensitive only to pretreatment blockade. This would suggest that the maintenance of one class of caffeine-activated intracellular stores requires entry of Ca²⁺ through Cd²⁺-sensitive Ca²⁺ channels. This differential temporal requirement for Ca²⁺ channels in Ca²⁺ signaling may be a mechanism to segregate intracellular signaling pathways of multiple neuroendocrine regulators in the teleost pituitary.