The present study examined the changes in mRNA levels of a pituitary-specific trans-acting factor, Pit-1, and prolactin during the rat estrous cycle. Total cytoplasmic RNA was analyzed by Northern blot and slot-blot hybridization to examine the prolactin mRNA level. Reverse transcription-polymerase chain reaction (RT-PCR) was performed to examine the Pit-1 mRNA level. Proestrous and estrous prolactin mRNA levels were significantly higher than the metestrous and diestrous levels, whereas Pit-1 mRNA levels of the estrous and metestrous stages were about two- to threefold higher than those of the proestrous and diestrous stages. Proestrous Pit-1 mRNA levels increased gradually from 10.00 h to 20.00 h, while prolactin mRNA levels slightly decreased until 14.00 h but increased later until 20.00 h. During the rat estrous cycle, especially in the afternoon of the proestrous day, changes of prolactin mRNA levels seem to follow a prior increase of Pit-1 mRNA. Therefore, Pit-1 may be partly involved in the regulation of prolactin gene expression according to the rat estrous cycle. Estradiol administration to ovariectomized rats significantly increased both the mRNA levels of prolactin and Pit-1, which suggests that the gene expression of Pit-1 is regulated by estrogen through indirect extracellular pathways.