The acute effects of a single injection of perphenazine on pituitary and blood prolactin levels were studied in male rats and rabbits. Prolactin from both sources was determined by bioassay. A significant rise in blood prolactin became visible as early as 0.5 h after injection of perphenazine – earlier times were not studied – and remained high for at least 4 h. The prolactin peak following intravenous injection in rabbits was attained after 1 h, while in rats injected sub-cutaneously, it was reached after 2 h. Concomitantly, pituitary prolactin in rats decreased, falling to a minimum level within 2 h, and remaining low for at least 4 h. These results are interpreted as further evidence that perphenazine induces immediate release of prolactin from the pituitary into the blood. This release is probably due to suppression of the hypothalamic prolactin-inhibiting factor. The possibility that perphenazine promotes prolactin synthesis is also discussed.