Although estrogen is known to stimulate the secretion of prolactin, there are only slight differences between the prolactin levels in the follicular and luteal phases in normal women. To test the hypothesis that progesterone is involved in the regulation of prolactin release, 50 mg of progesterone was administered intramuscularly at 0600 h to twelve hypogonadal women and blood samples were obtained at 15 min intervals between 1500 and 2000 h to determine the prolactin levels. The day before progesterone treatment, control blood samples were obtained at 15 min intervals between 1500 and 2000 h. The serum progesterone levels were 28.7 +/- 4.1 ng/ml at 1500 h, 24.2 +/- 3.5 ng/ml at 1730 h and 21.3 +/- 2.9 ng/ml (mean +/- SD) at 2000 h. In eight of twelve hypogonadal women, progesterone lowered circulating prolactin levels significantly. These results indicate that a high level of progesterone in the luteal phase may partly block estrogen-induced prolactin release physiologically.