The in vitro assay of colony forming unit-erythroid (CFU-E) colony is a direct and most reliable method to evaluate a biological activity of erythropoietin (EPO). In general, two kinds of cells are employed in this assay system as an effector cell, e.g. bone marrow cell and fetal mouse liver cell, and the latter cell has more sensitivity for EPO. In this paper, a fundamental methodological process in vitro CFU-E colony formation using a fetal mouse liver cell was described in detail and sensitivity for EPO as well as reproducibility of formed colony count were checked up. In addition, an inhibitory effect was indicated in plasma, even though sampled from a healthy volunteer and it was confirmed to be eliminated sufficiently by simple pretreatment with acidification combined with boiling.