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 microliters) 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 less than 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 less than 0.01), whereas higher doses (1 microgram, 0.74 nmol, or 5 micrograms, 3.71 nmol) had a stimulatory effect on PRL release (p less than 0.01). Similarly, a low dose of SP (0.1 microgram, 0.07 nmol) injected intravenously lowered plasma PRL (p less than 0.05). Large doses of intravenous SP (50 micrograms, 37.1 nmol) dramatically stimulated PRL release (p less than 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.(ABSTRACT TRUNCATED AT 250 WORDS)