Estrogens are recognized regulators of the expression of neurohypophyseal hormones and of anterior pituitary prolactin (PRL). Here we have investigated whether the levels of PRL mRNA and of 23 and 14 kDa PRL variants present in the hypothalamo-neurohypophyseal system change during the estrous cycle or in response to estrogen treatment. The reverse transcription polymerase chain reaction (RT-PCR) was performed to examine PRL mRNA expression in isolated paraventricular (PVN) and supraoptic (SON) hypothalamic nuclei. In both nuclei PRL mRNA levels appeared higher in cycling females than in male rats, with the highest level occurring at estrus. This increase may involve estrogen action, since estrogen administration to ovariectomized rats was associated with apparently higher PRL mRNA levels in both the PVN and SON. Expression of the PRL gene at these sites may occur via both transcriptional factor Pit-1-dependent and -independent mechanisms. RT-PCR detected the mRNA for Pit-1 in the PVN but only at estrus. The concentration of the 23 kDa immunoreactive PRL determined in the neurohypophysis was significantly higher during estrus and after estrogen treatment. However, no difference was detected in the levels of the neurohypophyseal 14 kDa PRL-like fragment along the estrous cycle nor after estrogen administration. This lack of parallelism between neurohypophyseal PRLs could relate to an estrogen-induced inhibition of the proteolysis of 23 kDa PRL at this site, since estrogen treatment reduced the activity of neurohypophyseal proteolytic enzymes able to cleave PRL. Altogether our results are consistent with estrogens having a stimulatory effect on PRL gene expression in the hypothalamo-neurohypophyseal system and a concomitant inhibitory action on PRL proteolysis at this site.