In vitro erythropoiesis inhibitory activity (EIA) was isolated and partially purified from sera obtained from a patient with chronic renal failure (CRF). EIA was purified from human sera by ammonium sulfate precipitation, column chromatography and PAGE electophoresis, and the corresponding biological activity was assayed in erythroid colony (CFU-E) cultures. Ammonium sulfate precipitation revealed that the EIA resided in two high molecular weight fractions which yielded a single peak on G-200 Sephadex. PAGE electrophoresis demonstrated that the EIA fraction separated into 3 bands with a molecular weight range of 12,000–50,000 daltons. Antibody to EIA was prepared in rabbits, and Ouchterlony immunodiffusion assays employing the antibody material revealed that several CRF sera generated intense double preciptin bands when tested in the assay. In contrast, normal sera consistently yielded a weak preciptin band. These results suggest the presence of reduced amounts of EIA-like material in normal humans and elevated levels in CRF sera. The significance of EIA in the anemia of CRF is discussed.