Prolactin, via its receptor variants, plays an important role in various tissues including brain. Many studies have been performed to define the regulation of prolactin and its receptor in the pituitary gland, both in females and males of different species. However, gender differences at transcriptional level have not been completely established nor has its expression in different brain regions. The aim of this study was to determine expression of prolactin and its receptor in the pituitary of male and female rats during the estrous cycle, and compare it with the expression regulation in selected brain areas such as hypothalamus and hippocampus. Quantitative polymerase chain reaction (PRC) was performed to assess expression of prolactin and its receptor. The results indicate that prolactin and its receptor are more abundant in the pituitary gland of female rats, and it varies during the estrous cycle, presenting its maximal expression on proestrus day. Interestingly, in hypothalamus and hippocampus, prolactin expression was more abundant in male than in female rats and did not vary significantly during the estrous cycle. However, prolactin receptor was consistently more abundant in female tissues in all studied regions, suggesting that females have more availability of prolactin receptor for physiological functions. The overall results indicated that prolactin and its receptor have a differential expression regulation in male and female rats, suggesting a sexual dimorphic role of prolactin in pituitary, hypothalamus, and hippocampus.