Erythropoiesis stimulating agents (ESAs) have been reported to activate erythropoietin receptors (EpoR) on cell types, including endothelial, neuronal, renal tubule, and cardiac cells. ESAs have also been reported to promote angiogenesis. However, those findings are controversial and confounded by methodologic issues. We show that EpoR mRNA was detected in essentially all cell types examined, including primary human endothelial, renal, cardiac, and neuronal cells but 10- to 100-fold lower than Epo-responsive cells using quantitative reverse-transcribed polymerase chain reaction. Total endothelial EpoR protein examined using a new monoclonal antibody was low to undetectable. Surface EpoR on endothelial cells was not detected using [(125)I]-rHuEpo surface-binding studies. There was no evidence of ESA-induced intracellular signaling in endothelial cells. There was a similar lack of EpoR expression and signaling in other cell types examined. Experiments were performed examining ESA function on these cells. An in vivo rat corneal angiogenesis assay demonstrated neo-vessel formation in response to recombinant human vascular endothelial growth factor (rHuVEGF). However, recombinant mouse Epo did not induce vessel formation. Similarly, ESAs did not reproducibly provide cytoprotection to neuronal, renal, or cardiac cells. Taken together, our data challenge the notion of presence or function of EpoR on nonhematopoietic cells, and call into question the preclinical basis for clinical studies exploring direct, "pleiotropic" actions of ESAs.