Methods for the cultivation of erythroid colonies in vitro are expected to allow selective assay of the earliest committed erythropoietic progenitors in the hemopoietic tissues of man and other species. In the present study, factors affecting erythroid colony formation were examined in methyl cellulose cultures of mouse bone marrow. Efficiency of colony formation observed after 2 days of culture was increased as much as 5-fold (to an average of 325 colonies/10-5 nucleated marrow cells) by the addition of thiol (either beta-mercaptoethanol or alphal-thioglycerol) at a final concentration of 10 minus 4 M. Optimum efficiency required 0.5 erythropoietin units/ml and was influenced by the purity of the preparation. When cultures contained thiol and high doses (3 units/ml) of purified erythropoietin, a second population of erythroid colonies became apparent after 5 days of culture, and increased in size to macroscopic dimensions by the tenth day, when they contained as many as 10-4 cells. Feeding was not required. These colonies, thought to be analogous to the "bursts" reported by Axelrad and coworkers in plasma clot cultures, were observed here at a 6-fold higher frequency (25/10-5 marrow cells) and were linearly related to the number of marrow cells plated, down to limiting numbers of colonies. On the basis of their impressive proliferative potential exhibited in culture, the cells originating these colonies are thought to represent a class of very early erythropoietin-responsive red cell progenitors.