Erythropoietin (EPO) is the cytokine essential for erythropoiesis; however, the expression of EPO and EPO receptor (EPOR) is not limited to cells of erythroid lineage. Erythropoietin and EPOR expression has been localized to numerous non-hematopoietic cells and tissues including endothelial, neuronal and ovarian. Moreover, several tumor types have been shown to express both EPO and EPOR and display increased expression upon hypoxia. To shed light on the potential biological role of EPOR and tumor cells we characterized the response of human colon carcinoma HT-29 cells to EPO. Functionality of the erythropoietin receptor was assessed by radio-labeled ligand binding, cellular proliferation/signaling, and gene expression using DNA microarrays. Receptor binding experiments using [125I]-EPO did not reveal measurable EPO binding activity present on the surface of HT-29 cells under both normoxic and hypoxic conditions. Moreover, EPO failed to induce cellular proliferation or an increase in the phosphorylation state of STAT5, EPOR or ERK1/2 under normoxic or hypoxic conditions at supra-pharmacological levels (25 IU/ml). Gene expression analysis revealed no significant change in gene expression in response to EPO (5 IU/ml) under normoxic conditions. On the other hand, over 347 genes exhibited greater than a 1.5 fold change in gene expression when cells were cultured under hypoxic conditions (1 % O2). When EPO was administered to cells in the hypoxic state, 36 additional genes were observed (9 and 27 up-regulated or down-regulated, respectively). That HT-29 cells exhibit minor transcriptional changes in response to EPO raises the possibility that EPO may signal in HT-29 cells. However, the mechanism for this response is not through the previously described EPO/EPOR signal transduction pathway. This conclusion is supported by the apparent lack of EPO receptor expression on the cell surface. These results suggest that tumor microenvironment, e.g., hypoxia, exerts a greater effect than that seen by exposure to erythropoietin.