Prolactin (Prl) receptor (Prlr) gene is expressed in various brain regions, with the highest level present in the choroid plexus, a site for receptor-mediated PRL transport from the blood to cerebrospinal fluid. We investigated the regulatory mechanism of Prlr gene expression by PRL in the murine choroid plexus. We first examined the organization of the alternative first exons in murine Prlr gene. In addition to the three known first exons, mE1(1), mE1(2), and mE1(3), two first exons, mE1(4) and mE1(5), were newly identified by cDNA cloning. Each first exon variant of Prlr mRNA exhibited tissue-specific or generic expression. In the choroid plexus of mice, the expression levels of mE1(3)(-), mE1(4)(-), and mE1(5)(-)Prlr mRNAs were increased in the lactating mice compared with those in the diestrus mice. Furthermore, the expression level of mE1(4)-Prlr mRNA was decreased in the PRL-deficient (Prl (/) ) mice compared with the PRL-normal (Prl (+/-/+/-) and Prl (+/-/-)) mice. In the ovariectomized Prl(-/-) mice, the expression level of mE1(4)-Prlr mRNA was significantly increased by PRL administration but not by 17 beta-estradiol administration. The expression levels of the two last exon variants of Prlr mRNAs, encoding the long and short cytoplasmic regions of PRLR, were also increased in the lactating mice and decreased in the Prl(-/-) mice. These findings suggest that PRL stimulates the Prlr gene expression through the transcriptional activation of mE1(4) first exon, leading to increases in the long-and short-form variants of Prlr mRNA in the murine choroid plexus. Journal of Molecular Endocrinology (2012) 48, 169-176