Prolactin plays an essential role in the development of rodent mammary tumors and is a potent mitogen in human normal and cancerous breast tissues/cells. In this study, we have analyzed the expression of prolactin receptors, including the long receptor form (LF; stimulatory) and two novel short forms (SFs; S1a and S1b) derived from alternative splicing that are inhibitory of the activation induced by prolactin through the LF. Southern analysis of breast cancer profiling arrays revealed that 29 patients (group I) expressed an elevated LF, 10 patients (group II) showed decreased LF, and 8 patients (group III) had no change relative to the adjacent normal tissue. Their respective SF expression was increased in 21 patients of group I and generally decreased in groups II and III. However, the ratio of SF to LF was significantly decreased in 76% of the breast tumors and distributed evenly among the groups. Quantification of differential expression of prolactin receptor variants by real-time PCR in 15 pairs of human normal and tumor breast-matched tissues revealed a similar significant decrease in the ratio of SF to LF in the tumor tissue. Consistent lower ratio of SFs to LFs was confirmed in 8 of ten different breast cancer cell lines compared with normal mammary Hs578Bst and MCF10A cells. Because SFs act as dominant negative regulators of the stimulatory actions of the LF in vitro, their relatively reduced expression in cancer could cause gradations of unopposed prolactin-mediated LF stimulatory function and contribute to breast tumor development/progression.