Serum prolactin levels were determined by radioimmunoassay in hypophysectomized, ovariectomized rats bearing 0, 1, 2, or 4 anterior pituitaries (AP) underneath the kidney capsule at 1, 2, 4, 6, 8, and 10 weeks after transplantation. Rats with no AP transplants had barely detectable levels of serum prolactin, whereas rats with 1 AP transplant from female cycling rats had serum prolactin values as high as those seen in estrous rats (120 ng/ml serum). In rats bearing 2 AP transplants, serum prolactin increased to 170 ng/ml serum, whereas 4 AP transplants elevated serum prolactin to 250 ng/ml, which is about equivalent to that in lactating postpartum rats (280 ng/ml). Injections of estradiol benzoate (1 µg/day for 5 days) into rats bearing 0, 1, 2, or 4 AP transplants, beginning 10 weeks after transplantation, increased serum prolactin over pre-treatment levels, except in the rats with no pituitary transplants. Bilateral lesions placed in the median eminence or anterior hypothalamus of ovariectomized rats significantly increased serum prolactin to 125 and 85 ng/ml, respectively, as compared with sham-lesioned controls (20 ng/ml). Posterior hypothalamic lesions increased serum prolactin concentration slightly and lesions in the amygdaloid nuclei had no effect. These results indicate that removal of hypothalamic inhibition of prolactin release by pituitary transplantation or by appropriate lesion placement in the hypothalamus results in elevated serum prolactin levels.