A time course study of the sequential appearance of erythropoietin-dependent colonies and bursts (derived from CFU-E and BFU-E, respectively) was performed on mouse hemopoietic cells cultured in methyl cellulose containing 2-mercaptoethanol. A new type of small, short-lived burst was found to be apparent by the third day in culture. By the sixth day most of these bursts had lysed. At the same time, differentiating erythroblasts began to be detectable in the large, late appearing bursts described previously. These two types of burst, differing from each other and from CFU-E derived colonies both in their ultimate size and morphology, as well as in their time course of appearance and lysis, were compared in other ways. It was found that early burst formation required about 100 times more erythropoietin than that needed to stimulate CFU-E. On the other hand, early burst formation required less than one-quarter of the amount of erythropoietin needed to obtain the large, late appearing bursts. Comparison of the distribution of early burst progenitors relative to pluripotent stem cells (CFU-S) in individual spleen colonies gave a correlation coefficient that was also intermediate between that obtained comparing CFU-S with CFU-E and that obtained comparing CFU-S with the progenitors of late bursts. These results suggest that decreasing proliferative capacity is associated with progressively increasing erythropoietin responsiveness as primitive erythropoietic progenitors move from a position close to pluripotent stem cells through several differentiation steps to reach a stage just prior to the onset of detectable hemoglobin synthesis.