SUMMARY The concentration of prolactin in serum after oestrogen and progesterone injection into spayed rats was measured by radioimmunoassay. After a single injection of 5 μg oestradiol benzoate (OB) into long-term ovariectomized rats, serum prolactin concentrations showed a circadian rhythm with high levels in the afternoon and almost no changes in the morning. Peaks of prolactin occurred 2, 3 and 4 days after the injection. Below a dose of 1 μg OB, the response was dose-dependent, but the response was then maximal. In spayed rats primed with 5 μg OB, the injection of 2 mg progesterone 2, 3 or 4 days later resulted in a significant increase in serum prolactin. This response, in contrast to that of oestrogen, occurred in the morning and in the evening and was found to be dose-dependent. The rise in serum prolactin after injection of 1 mg progesterone also showed a close relationship to the priming dose of OB. Progesterone had no effect in spayed, untreated animals. Maximal levels of prolactin were attained 3–4 h after the s.c. injection of progesterone. The release of prolactin which can be induced either by OB or by progesterone was blocked by the administration of progesterone injected 1 day before the expected release would occur. These results indicate that progesterone exerts both facilitatory and inhibitory effects on prolactin secretion. Male rats were found to be less sensitive to the ovarian steroid treatment. It is suggested that oestrogen could be responsible for the rise in prolactin observed at pro-oestrus and progesterone for the increase in prolactin in pseudopregnancy and pregnancy.