In order to examine the hormonal regulation of the prolactin-receptor (PRL-R) gene expression during mammary gland development, ewes were treated to induce lactation via an estrogen-progesterone-hydrocortisone and ovine growth hormone treatment. In situ hybridization analysis was used and revealed that sex steroids increased PRL-R mRNA levels in the mammary gland. Using RNase protection assay we showed that the estradiol + progesterone treatment increased both the levels of the long and the short forms of PRL-R mRNA. Addition of hydrocortisone increased the level of alphaS1-casein transcripts and the level of the ratio of the long to the short form of the PRL-R mRNA. This ratio can be further enhanced by addition of ovine growth hormone to the latter treatment. This suggests a role of hydrocortisone and ovine growth hormone in the alternative splicing that leads to the preferential expression of the long form of the PRL-R mRNA. In conclusion, the present experiments suggest that estrogen, progesterone and hydrocortisone are the major regulators of the PRL-R gene expression during pregnancy and prepare the mammary gland for its differentiation.