Neurotensin (NT) is a potent stimulator of electrical and secretory activities in frog pituitary melanotrophs. The aim of the present study was to characterize the transduction pathways associated with activation of NT receptors in frog melanotrophs. Application of synthetic frog NT (f NT) increased the cytosolic calcium concentration ([Ca²⁺]_c) and stimulated the formation of inositol trisphosphate (IP₃). The phospholipase C inhibitor U-73122 blocked the electrophysiological and secretory effects of f NT. Intracellular application of the IP₃ receptor antagonist heparin abolished f NT-induced electrical activity. Suppression of Ca²⁺ in the incubation medium markedly reduced the effect of NT on [Ca²⁺]_c, firing rate and -MSH secretion. Similarly, the inhibitor of IP₃-induced Ca²⁺ release and store-operated Ca²⁺ channels 2-APB, and the non-selective Ca²⁺ channel blockers GdCl₃ and NiCl₂, attenuated the [Ca²⁺]_c increase and the electrical and secretory responses evoked by f NT. Co-application of the L- and N-type Ca²⁺ channel blockers nifedipine and -CgTx GVIA reduced the effects of f NT on action potential discharge, [Ca²⁺]_c increase and -MSH release. The PKC inhibitors PKC (19-31) and chelerythrine reduced the electrophysiological and secretory responses induced by iterative applications of f NT. Collectively, these results demonstrate that, in frog melanotrophs, neurotensin stimulates the PLC/PKC pathway and increases [Ca²⁺]_c. Both Ca²⁺ release from intracellular stores and Ca²⁺ influx through L- and N-type Ca²⁺ channels, are involved in fNT-induced -MSH secretion. In addition, the present data indicate that PKC plays a crucial role in the maintenance of the responsiveness of melanotrophs to NT.