Critical studies were made on erythroid colony formation from cultured fetal mouse liver cells in an attempt to develop a simple and sensitive erythropoietin (Epo) assay procedure. The maximum colony formation was observed 24 h after plating of the cells when an evident dose-response relation was found for Epo added. The colony forming ability decreased steadily as the gestational age of the fetus advanced and was gradually lost by postnatal days 10-11. By morphological and cytochemical criteria almost all the colonies were found to be erythroid. 59Fe-labelling experiments revealed a fairly good correlation between the colony number and 59Fe incorporation into both cells and haem. Dose-response curves for plasma were parallel to the Epo standard curve. Based on these findings we developed a procedure which could measure as little as 0.4 mU of Epo without requiring 59Fe. Using this method, plasma Epo titres were determined in 16 normal and 69 anaemic subjects.