Reduced expression of the proto-oncogene c-myb appears necessary for erythroid differentiation induced by chemical agents and by the natural regulator, erythropoietin (Epo). Treatment of Epo-responsive Rauscher erythroleukemia cells with an anti-sense oligodeoxynucleotide to c-myb in the absence of other inducers downregulated myb protein markedly and caused hemoglobinization of the cells within 48 hours. Epo treatment, which downregulates c-myb in these cells, induced hemoglobinization to the same degree. Epo also induced the appearance of anion transport protein on the plasma membrane, consistent with terminal differentiation. In contrast, antisense c-myb did not induce this erythroid marker. The results are consistent with a role for myb protein in the regulation of hemoglobin synthesis.