The primary structure of frog neurotensin ( fNT) has recently been determined and it has been shown that fNT is a potent stimulator of α-MSH secretion by frog pituitary melanotropes. In the present study, we have investigated the effects of fNT on the electrical activity of cultured frog melanotropes by using the patch-clamp technique and we have determined the pharmacological profile of the receptors mediating the effect of fNT. In the cell-attached configuration, fNT (10<sup>–7</sup> M) provoked an increase in the action current discharge followed by an arrest of spike firing. In the gramicidin-perforated patch configuration, fNT (10<sup>–7</sup> M) induced a depolarization accompanied by an increase in action potential frequency and a decrease in membrane resistance. Administration of graded concentrations (10<sup>–10</sup> to 10<sup>–6</sup> M) of fNT or the C-terminal hexapeptide NT(8–13) caused a dose-dependent increase in the frequency of action potentials with EC<sub>50</sub> of 2 × 10<sup>–8</sup> and 5 × 10<sup>–9</sup> M, respectively. The stimulatory effect of fNT was mimicked by various pseudopeptide analogs, with the following order of potency: Boc-[Trp<sup>11</sup>]NT(8–13) > Boc-[ D-Trp<sup>11</sup>]NT(8–13) > Boc-[Lys<sup>8,9</sup>, Nal<sup>11</sup>]NT(8–13) > Boc-[Ψ11,12]NT(8–13). In contrast, the cyclic pseudopeptide analogs of NT(8–13), Lys-Lys-Pro- D-Trp-Ile-Leu and Lys-Lys-Pro- D-Trp-Glu-Leu-OH, did not affect the electrical activity. The NTS1 receptor antagonist and nts2 receptor agonist SR 48692 (10<sup>–5</sup> M) stimulated the spike discharge but did not block the response to fNT. In contrast, SR 142948A (10<sup>–5</sup> M), another NTS1 receptor antagonist and nts2 receptor agonist, inhibited the excitatory effect of fNT. The specific nts2 receptor ligand levocabastine (10<sup>–6</sup> M) had no effect on the basal electrical activity and the response of melanotropes to fNT. In cells which were dialyzed with guanosine-5′- O-(3-thiotriphosphate) (10<sup>–4</sup> M), fNT caused an irreversible stimulation of the action potential discharge. Conversely, dialysis of melanotropes with guanosine-5′- O-(2-thiodiphosphate) (10<sup>–4</sup> M) completely blocked the effect of fNT. Pretreatment of cells with cholera toxin (1 µg/ml) or pertussis toxin (0.2 µg/ml) did not affect the electrical response to fNT. Intracellular application of the G<sub>o/i/s</sub> protein antagonist GPAnt-1 (3 × 10<sup>–5</sup> M) had no effect on the fNT-evoked stimulation. In contrast, dialysis of melanotropes with the G<sub>q/11</sub> protein antagonist GPAnt-2A (3 × 10<sup>–5</sup> M) abrogated the response to fNT. The present data demonstrate that fNT is a potent stimulator of the electrical activity of frog pituitary melanotropes. These results also reveal that the electrophysiological response evoked by fNT can be accounted for by activation of a G<sub>q/11</sub>-protein-coupled receptor subtype whose pharmacological profile shares similarities with those of mammalian NTS1 and nts2 receptors.