Mammary gland development and hyperprolactinemia of rats were induced by estradiol (E2) treatment. The condition of the animals resembled that of during lactation. When it focused on high PRL levels, we studied the molecular phenotype of the rat mammary gland following E2 treatment, targeting prolactin (PRL) mRNA, using the sensitive techniques of solution and in situ reverse transcriptase-polymerase chain reaction (RT-PCR). In addition, we compared the PRL mRNA and plasma PRL level with those in the mammary glands of both pregnant and lactating rats. In the E2-treated groups, the plasma PRL levels were increased. No mammary PRL mRNA was detected in E2-treated rats by either solution or in situ RT-PCR. The plasma PRL level was extremely high and associated with the mammary gland development in lactating rats. In addition, mammary PRL mRNA was detected in both pregnancy and lactation by solution and in situ RT-PCR. These results suggested a lack of PRL mRNA in the mammary gland of E2-treated rats, despite similar plasma PRL levels and morphological changes to those in lactating rats. The induction of PRL mRNA coincident with the development of mammary gland in the pregnant and lactating rats, differed from that in the E2-treated rats in molecular biology, particularly in PRL mRNA expression, and suggested that PRL mRNA in mammary gland contributes to de novo synthesis of PRL in milk which is under the multihormonal regulation.