Despite the important role of prolactin (PRL) in mammary gland development and function, little is known about the distribution of the different forms of the prolactin receptor (PRLR) under various physiological circumstances. Here, the distribution of the long (LF) and the short (S3 in mouse) receptor common to both mice and rats was determined by immunofluorescence on frozen sections of virgin, pregnant and lactating mouse mammary gland. Myoepithelial cells were consistently and intensely stained for both receptors. For luminal cells at all stages (ducts and alveoli), a large proportion of PRLR staining was unexpectedly present on the apical face. In the non-lactating state, no basal staining of luminal cells was detectable. During lactation, a proportion of both receptors moved to the basolateral surface. In vitro, HC11 cells showed constitutive expression of LF but expression of S3 only upon the formation of adherent junctions. Tight junction formation was accelerated by incubation in pseudo-phosphorylated PRL, as measured by transepithelial resistance and the expression and placement of the tight junction protein, zonula occludens-1. Once an intact monolayer had formed, all LF and S3 receptors were apical (akin to the non-lactating state) and only apical application of PRL activated the Jak2-STAT5 and ERK pathways. By contrast, basolateral application of PRL resulted in a reduction in basal ERK phosphorylation, suggesting an involvement of a dual specificity protein phosphatase. Normal human breast samples also showed apical PRLRs. These results demonstrate important contextual aspects of PRL-PRLR interactions with implications for the analysis of the role of PRL in breast cancer.