Prolactin (PRL) is an important hormone in mammary tumorigenesis in rodents but its involvement in human breast cancer has been controversial. A role for locally produced PRL in breast carcinogenesis is suggested by its mitogenic action on breast cancer cells and the expression of both PRL and its receptor (PRL-R) in breast carcinomas. Our objective was to examine whether PRL, overexpressed by breast cancer cells, forms an autocrine/paracrine loop that confers a growth advantage for tumors. MDA-MB-435 breast cancer cells overexpressing 23K human PRL were generated, and PRL production and secretion by the clones were confirmed by RT-PCR, western blotting, and the Nb2 bioassay; control clones contain vector only. In vitro the 23K PRL clones proliferated faster and expressed higher levels of the PRL-R protein than controls only when incubated in charcoal-stripped serum (CSS) devoid of lactogenic hormones. When injected into the mammary fatpad of female nude mice or subcutaneously into males, the PRL-overexpressing clones formed tumors that grew 2-4-fold faster than tumors derived from control clones or wild type MDA-MB-435 cells. Western analysis demonstrated significantly higher PRL, PRL-R, and bcl-2 levels in the tumors overexpressing PRL compared to control tumors. These data support a role for breast PRL as a growth/anti-apoptotic factor and suggest that it may serve as a novel therapeutic target for the treatment of breast cancer.