EPO stimulates formation of red blood cells by binding to and activating the EPO receptor (EPOR) expressed on the surface of erythroid progenitor cells. Recently EPOR was reportedly expressed in other, non-hematopoietic tissues including brain, kidney, heart and endothelium. EPOR expression has also been reported in tumor tissues and tumor cell lines and many investigators have suggested that EPO may promote tumor growth. This has raised concerns about the use of rHuEPO to treat anemia associated with chemotherapy in cancer patients. To evaluate this, we have examined EPOR expression and binding in tissues and cell lines by quantitative PCR, Western blot analysis and EPO binding assays. Quantitative RT-PCR analysis revealed that the EPOR gene is transcribed in most tissues and cell lines. However non-hematopoietic EPOR transcript levels were lower than that found in hematopoietic cells. No evidence of elevated EPOR gene transcription in tumor vs. non-tumor tissues or in tumor cell lines analyzed was observed. Using Western analysis, we found that commercial anti-EPOR antibodies commonly used to examine EPOR protein expression in published tumor studies, cross-reacted with multiple proteins and/or reacted with proteins of sizes different from that predicted for EPOR. This suggests that in many publications where these antibodies were used, claims of expression of EPOR protein in tumors are of questionable significance. A rabbit polyclonal antibody was developed that specifically reacted with a protein band on Western blots of the size predicted for EPOR. The antibody reacted specifically with a band in COS cells transfected with a human EPOR expressing construct but not in transfection controls. This antibody also reacted with the same sized protein in non-hematopoietic cells. We concluded that EPOR mRNA and EPOR protein were present in most normal cell types examined. Thus the detection of EPOR in tumors and tumor cell lines likely represents the continued expression reflecting the tissue of tumor origin. Since expression does not equate to the presence of surface receptor, binding assays with 125I EPO to non-hematopoietic tumor cell lines were performed. Tumor cell lines showed weak to no detectable EPO binding. These results suggest that although EPOR protein is synthesized in these cells, little or no receptor is expressed on their cell surface. Furthermore, if limited amounts of EPO bound EPOR in tumor lines any biological effect may be different or muted compared to classical responses in hematopoietic cells.