It was discovered that monoclonal anti-erythropoietin (EPO) antibody CFC-6 can neutralize EPO-induced cell activation. To know the binding site of CFC-6, recombinant human erythropoietin (rhEPO) was digested with Glu-C, followed by a separation using high performance liquid chromato graphy (HPLC). Each HPLC fraction was blotted on the nitrocellulose membrane and the membrane was treated with anti-EPO antibody CFC-6 and anti-mouse antibody which is modified with peroxidase. Only one spot showed the color and the fraction was sequenced by Edman degradation. The results suggest that CFC-6 recognizes amino acid sequence V63-W-Q-G-L-A-L-L-S-E72 which is a part of helix II of the EPO molecule. Binding of CFC-6 to EPO may inhibit EPO binding to its receptor, which implies that the antibody binding site and the receptor binding site are close or overlapping.