To evaluate a possible physiological role of endogenous substance P (SP) in the control of prolactin (PRL) release, conscious adult male rats were given injections of a specific antiserum against SP (anti-SP) into the third ventricle (3 µl) or intravenously (0.5 ml). Third-ventricular injection of anti-SP induced a significant increase in plasma PRL levels when compared to values in control animals injected with normal rabbit serum (p < 0.02). Plasma PRL concentrations were significantly elevated within 2 h after injection of antiserum and remained elevated for the 4-hour duration of the experiment. In contrast, injections of large doses of anti-SP intravenously had no effect on plasma PRL levels. In order to confirm the effect of SP itself, synthetic SP was injected intravenously and intraventricularly. Opposite effects of SP on PRL release were observed after intravenous and intraventricular injections of low or high doses of the peptide. A lower dose of SP (10 ng, 7.42 pmol) injected into the third ventricle suppressed the release of PRL (p < 0.01), whereas higher doses (1 µg, 0.74 nmol, or 5 µg, 3.71 nmol) had a stimulatory effect on PRL release (p < 0.01). Similarly, a low dose of SP (0.1 µg, 0.07 nmol) injected intravenously lowered plasma PRL (p < 0.05). Large doses of intravenous SP (50 µg, 37.1 nmol) dramatically stimulated PRL release (p < 0.001). To evaluate a possible direct action of SP on PRL release from the anterior pituitary, the peptide was incubated with dispersed anterior pituitary cells for 1 h. The release of PRL from incubated anterior pituitary cells was not affected at any dose of synthetic SP tested (10<sup>–9</sup>–10<sup>–6</sup> M. These data indicate that in the male rat endogenous SP is a physiologically significant inhibitor of basal release of PRL, via a hypothalamic action, although higher, presumably pharmacological doses of SP stimulate the release of this hormone also via hypothalamic action.