The production of recombinant human erythropoietin (r-HuEPO) began with a search for the gene coding for human erythropoietin (EPO). Two radiolabeled pools of oligonucleotide probes were designed, based on amino acid sequence information obtained from human urinary EPO. Each probe, consisting of complex mixtures of 128 short synthetic sequences of DNA, was used to search a human genomic library for clones containing the human EPO gene sequence. To verify that the isolated clones contained the complete functional gene encoding human EPO, these sequences were expressed in Chinese hamster ovary cells, and the secreted r-HuEPO was purified, characterized, and compared with the human urinary hormone using a variety of different techniques. Results of these studies indicate that r-HuEPO is virtually indistinguishable from human urinary EPO in its biochemical and immunological properties. It is a 165-amino acid protein whose primary sequence is identical to that of urine-derived EPO. The subsequent development of large-scale cell culture and production techniques has made available sufficient amounts of r-HuEPO for clinical use in the treatment of the debilitating anemia that almost invariably accompanies chronic renal failure.