The differential, tissue-specific regulation of oxytocin (OT) binding sites allows the neurohypophysial nonapeptide OT to fulfill a dual role: to induce uterine contractions at parturition and to mediate milk ejection during lactation. Whereas uterine OT binding sites are up-regulated prior to parturition and are rapidly down-regulated thereafter, mammary gland OT binding sites gradually increase throughout gestation and remain up-regulated during the ensuing lactation period. Here, we structurally characterized OT receptor (OTR) mRNA in mammary gland and analyzed its expression during gestation and lactation and in response to steroid treatment. In mammary gland tissues, we found a 6.7 and a 5.4 kb OTR mRNA species, and both species were further analyzed by RACE (rapid amplification of cDNA ends). The 6.7 kb mRNA was found to be common to mammary gland and uterus and to extend 618 nucleotides beyond the published sequence of the rat OTR gene. The 5.4 kb mRNA species is unique to the mammary gland and terminates at a mammary gland-specific polyadenylation site that is not preceded by a classical polyadenylation signal. RT-PCR analysis did not provide any evidence for differences in the coding regions, suggesting that both uterine and mammary gland OTR mRNAs encode the same receptor protein. Furthermore, primer extension experiments showed that no differences exist in the specific transcriptional initiation sites of the OTR gene in the two tissues. During pregnancy, OTR mRNA per mammary gland increased approximately 150-fold and remained high during lactation, consistent with the previously identified regulation of OT binding sites and the role of OT during lactation. Whereas estrogen administration strongly induced the uterine OTR mRNA levels (>5-fold), mammary gland remained unaffected by steroid treatment. Moreover, tamoxifen had no effect on the mammary gland OTR mRNA level. In summary, our data demonstrate a differential control of OTR expression in uterus versus mammary gland and a mammary gland-specific OTR mRNA polyadenylation site. However, this differential control apparently does not involve the expression of different receptor genes nor the utilization of tissue-specific transcriptional initiation sites.