The release of neurotensin by hypothalami from male rats was investigated in vitro using tissue fragments incubated in Krebs-Ringer bicarbonate-glucose buffer at 37 degrees C. Neurotensin was measured by radioimmunoassay using an antibody directed toward the C-terminal portion of the peptide. Neurotensin-like immunoreactivity released into the incubation medium eluted from Sephadex G-25 in a position identical to that of the synthetic peptide and serial dilutions of incubation medium were parallel to those of synthetic neurotensin in the radioimmunoassay. Neurotensin-like immunoreactivity released into the incubation medium was degraded during the incubation period by released hypothalamic peptidases. The addition of bacitracin (0.5 mg/ml) to the medium partially prevented this degradation. Neurotensin release was stimulated by dibutyryl cyclic AMP (10(-4) M) and by depolarizing concentrations of potassium. The latter effect was shown to be Ca2+-dependent. Dopamine (10(-4)--10(-6) M) stimulated neurotensin release in a dose-dependent manner and this effect was blocked by the dopamine receptor antagonist, haloperidol. Neurotensin release was not stimulated by either norepinephrine (10(-4) M) or serotonin (10(-4) M). The results indicate that neurotensin is released by the hypothalamus in vitro; its release is stimulated by membrane depolarization in a Ca2+-dependent manner and may involve an adenylate cyclase mechanism; and dopamine appears to serve as a stimulatory neurotransmitter for neurotensin-containing neurons.