Although prolactin has been shown to entrain hair growth cycles in seasonally responsive mammals, no comparable role has been identified in the age-dependent pelage replacement of rodents. Prolactin binds to dimerised membrane-associated prolactin receptors (PRLR) on target organs and initiates signalling via a number of intracellular pathways including the JAK-Stat5b pathway. The role of this pathway in murine hair growth was investigated by (i) PRLR gene disrupted mice, (ii) altered circulating prolactin profiles and (iii) impaired PRLR signal transduction with Stat5b gene-disrupted mice. The long (PRLR-L), and two short (PRLR-S2 and PRLR-S3) forms of the prolactin receptor were shown by RT-PCR to be expressed in the skin. These receptors were immunolocalised to the outer root sheath of the follicle as well as the epidermis and sebaceous gland. The first hair replacement cycle in PRLR gene-disrupted mice was advanced by 4 days in males and 2 weeks in females compared to wild type controls. Similarly, bromocriptine suppression of prolactin secretion advanced hair replacement by four days, which could be reversed by administration of exogenous prolactin at 18-22 days of age. In contrast, hair replacement in Stat5b-disrupted mice was delayed by two weeks. The duration of anagen, measured as the period of fibre elongation, did not differ between treated and control groups in any of these experiments. Pelage structure, fibre length and fibre diameter was not substantially altered. Pelage renewal across the body was slower during pregnancy and pseudopregnancy, and halted completely during lactation. Only after weaning was follicle reactivation resumed. As a key gestational and lactation hormone, prolactin is a likely candidate as a modulator of hair cycling at this time. An inverse relationship between PRLR-L mRNA and prolactin mRNA demonstrates the high level of prolactin signal regulation in the skin during reproduction. Hence, both reduced circulating prolactin levels and PRLR-deficiency results in a shorter telogen and hair renewal at a younger age. In contrast, when the signalling protein Stat5b is absent, follicle growth was retarded and new hair growth occurred later than in wild type mice. This could be explained by the altered pituitary feedback regulation of lactotrophs resulting in hyperprolactinemia in Stat5b-deficient mice. These results provide strong evidence that prolactin inhibits the activation of murine hair follicle growth.