Erythropoietin (EPO) is the principal hematopoietic cytokine that regulates mammalian erythropoiesis by binding to its transmembrane receptor EpoR. Recent experimental evidence suggests that the biologic effects of EPO are not limited to the regulation of erythropoiesis. In studies focusing on nonhematopoietic effects of EpoR signaling, we found high levels of EpoR protein expression in human breast cancer cells. The purpose of the present study was to evaluate clinical breast cancer specimens for EPO and EpoR expression, characterize the relationship between EPO expression and tumor hypoxia in biopsies prelabeled with hypoxia marker pimonidazole, analyze breast cancer cell lines for EpoR expression, and study the functional significance of EpoR expression in breast cancer cells in vivo. Immunohistochemical analysis for EPO, EpoR expression, and pimonidazole adducts was performed on 26 tumor biopsies with contiguous sections from 10 patients with breast cancer. High levels of EpoR expression were found in cancer cells in 90% of tumors. EPO expression was found in 60% of tumors and EPO and EpoR colocalization in tumor cells was present in many cases. The expression pattern of EPO with respect to tumor hypoxia was variable, without consistent colocalization of EPO and hypoxia in tumor cells. Human and rat breast cancer tissue culture cells express EpoR mRNA and protein. To study the in vivo function of EpoR expression in breast cancer cells, we used rat syngeneic R3230Ac mammary adenocarcinoma cells in a tumor Z-chamber model (dual porous plexiglass chambers containing fibrin gel, cancer cells, and a putative anti-tumor compound implanted into the subcutaneous tissue of rats). Local, one-time administration of a neutralizing anti-EPO antibody, soluble EPO receptor, or an inhibitor of Jak2, a cytoplasmic tyrosine kinase essential for EPO-mediated mitogenesis, resulted in a delay in tumor growth with 45% reduction in maximal tumor depth in tumor Z-chambers in a dose-dependent manner. These studies demonstrate the expression of functional receptors for EPO in breast cancer cells.