In amphibians, the secretion of α-MSH by melanotrope cells is stimulated by TRH and inhibited by NPY. We have previously shown that NPY abrogates the stimulatory effect of TRH on α-MSH secretion. The aim of the present study was to characterize the receptor subtypes mediating the action of NPY and to investigate the intracellular mechanisms involved in the inhibitory effect of NPY on basal and TRH-induced α-MSH secretion. Y₁ and Y₅ receptor mRNAs were detected by RT-PCR and visualized by in situ hybridization histochemistry in the intermediate lobe of the pituitary. Various NPY analogs inhibited in a dose-dependent manner the spontaneous secretion of α-MSH from perifused frog neurointermediate lobes with the following order of potency porcine peptide YY (pPYY) > frog NPY (fNPY) > porcine NPY (pNPY)-2–36) > pNPY-(13–36) > [d-Trp³²]pNPY > [Leu³¹,Pro³⁴]pNPY. The stimulatory effect of TRH (10⁻⁸6 m) on α-MSH release was inhibited by fNPY, pPYY, and [Leu³¹,Pro³⁴]pNPY, but not by pNPY-(13–36) and [d-Trp³²]pNPY. These data indicate that the inhibitory effect of fNPY on spontaneous α-MSH release is preferentially mediated through Y₅ receptors, whereas the suppression of TRH-induced α-MSH secretion by fNPY probably involves Y₁ receptors. Pretreatment of neurointermediate lobes with pertussis toxin (PTX; 1 μg/ml; 12 h) did not abolish the inhibitory effect of fNPY on cAMP formation and spontaneous α-MSH release, but restored the stimulatory effect of TRH on α-MSH secretion, indicating that the adenylyl cyclase pathway is not involved in the action of fNPY on TRH-evoked α-MSH secretion. In the majority of melanotrope cells, TRH induces a sustained and biphasic increase in cytosolic Ca²⁺ concentration. Preincubation of cultured cells with fNPY (10⁻⁷m) or ω-conotoxin GVIA (10⁻⁷m) suppressed the plateau phase of the Ca²⁺ response induced by TRH. However, although fNPY abrogated TRH-evoked α-MSH secretion, ω-conotoxin did not, showing dissociation between the cytosolic Ca²⁺ concentration increase and the secretory response. Collectively, these data indicate that in frog melanotrope cells NPY inhibits spontaneous α-MSH release and cAMP formation through activation of a Y₅ receptor coupled to PTX- insensitive G protein, whereas NPY suppresses the stimulatory effect of TRH on α-MSH secretion through a Y₁ receptor coupled to a PTX-sensitive G protein-coupled receptor.