An attempt was made to clarify the relationship between the synthesis of prolactin (PRL) and its secretion during the rat estrous cycle by examining the changes in PRL messenger RNA (mRNA); PRL secretion and anterior pituitary PRL content. At various stages of the estrous cycle, pituitary and serum levels of PRL and growth hormone (GH) were measured by radioimmunoassay, while their respective mRNA contents were determined by the hybridization method. The content of pituitary PRL mRNA did not increase in parallel with the increment of the serum level of the hormone. Significant increase in PRL mRNA were observed as three peaks; 1st peak at 14:00 on the proestrus (PE) day, 2nd peak at 4:00 and 3rd peak at 22:00 on the estrus (E) day. The GH levels in the pituitary and serum, or the change in PRL mRNA appeared to be unrelated to the rat estrous cycle. From the present results, we speculate that the three peaks of PRL mRNA content might be ascribable to the direct effect of estrogen on transcription, indirect activation of transcription followed by rapid and massive secretion of PRL, and mammotrope proliferation, respectively.