A new preparative method for isolation of human urinary erythropoietin (Ep) has been developed using hydrophobic interaction chromatography on Phenyl-Sepharose CL4B. Crude urine and urine concentrates from anemic patients were used directly without prior manipulation. In addition to facilitating hydrophobic interactions, Phenyl-Sepharose provided pi-pi interactions between its phenyl group on the gel matrix and the aromatic amino acid residues of Ep, and thus contributed to specific resolution. Over 90% of the urinary contaminants were excluded from the column, and Ep was selectively bound. Its activity was eluted with 20% ethylene glycol in 10 mM NaOH containing 4 M guanidine hydrochloride. This single step offered a mean purification factor of 110 with a recovery of 85%.