Lactating rats supplied with chronically indwelling intravenous (i.v.) and intraperitoneal (i.p.) catheters were allowed to suckle after a night of isolation from their offspring. The effect of infusions of saline and EDTA, given i.v. or i.p. on the suckling-induced rise of plasma prolactin was evaluated by radioimmunoassay. i.v. saline and EDTA did not affect the normal prolactin rise induced by suckling. However, i.p. EDTA completely blocked this rise, while the weight gain of the pups during the suckling period was only slightly depressed. i.p. EDTA had no suppressive effect on the perphenazine-induced rise of plasma prolactin and caused only a moderate inhibition of the prolactin rise in the plasma evoked by thyrotropin-releasing hormone and by brief exposure to ether. The experiments indicate, that because i.p. EDTA probably stimulates dopamine only slightly, it must inhibit prolactin secretion via another mechanism. This study, therefore, strongly supports the idea that more than one hypothalamic factors affect prolactin release during suckling.