A simple in vitro assay method for erythropoietin, a plasma glycoprotein which stimulates erythroid differentiation, has been developed. In this method, a liver cell suspension from 13-day mouse fetus was planted in the wells of a flat-bottomed 96-well microtiter plate and incubated for culturing. The formation of erythroid colonies, which were identified with benzidine staining, was dependent on added erythropoietin. Since this method is inexpensive, sensitive, simple, and only requires a small sample, it could be adopted for assaying the erythropoietin levels in blood, urine, and other materials.