A rapid spectrophotometric assay capable of detecting the hemoglobin content of 1000 mature erythrocytes has been utilized to quantitate the total hemoglobin synthesized by the progeny of circulating human erythroid progenitors in both the plasma clot and methylcellulose culture systems. The pronounced variation in the effect of different erythropoietin preparations on the hemoglobin content of cultured human peripheral blood bursts, previously described in a subjective manner, has been objectively quantitated. Further experiments demonstrated that both lymphocyte conditioned media and dexamethasone substantially increased the total hemoglobin synthesized by the progeny of cultured erythroid progenitors. The elevated amount of hemoglobin present in erythroid cultures containing either lymphocyte conditioned media and/or dexamethasone was due to both increased colony numbers and colony size. Assay of the total hemoglobin content per erythroid culture is an accurate, sensitive, measure of erythropoiesis in vitro and should be a valuable adjunct to the enumeration of BFU-E-derived erythroid colonies.