A rapid and simple chromatographic procedure has been developed for the large-scale purification of therapeutic-grade rHuEPO (recombinant human erythropoietin) from medium-conditioned cell cultures, which includes ion-exchange, hydrophobic-interaction and gel-filtration chromatography. A combination of these well-connected steps results in highly purified rHuEPO (> 99%), as revealed by SDS/PAGE and HPLC analyses, with a total yield of 38%. The specific activity of purified rHuEPO was 160,104 i.u./mg. Immunoblotting studies revealed that the protein possesses native EPO immunity. N-terminal sequencing of rHuEPO shows that the first 15 amino acids coincide with those of native EPO reported previously.