Erythropoietin (Epo) and dimethyl sulfoxide (DMSO) are believed to induce the differentiation of transformed erythroid cells by different signal transduction pathways. We have now obtained evidence for the interaction of these pathways. We used a Rauscher murine erythroleukemia cell line with a relatively low (8% to 10%) hemoglobinization response to Epo alone. Pretreatment of these cells for 1 day with DMSO followed by its removal and the addition of Epo resulted in a marked enhancement of the Epo specific hemoglobinization. We have designated this effect “DMSO priming.” This priming effect of DMSO on the Epo response was both time-dependent and DMSO concentration- dependent. DMSO priming potentiated the Epo response in three ways. Firstly, DMSO priming increased the total number of Epo responsive cells from 8% to 10% to 40% to 60%. Secondly, DMSO priming reduced the time required to reach the optimal Epo-induced response from 4 days to 2 days. Thirdly, the Epo dose-response curve was left-shifted approximately 20-fold. DMSO priming was also associated with a marked increase in Epo receptor density characterized by an apparently new receptor population and by the appearance of positive cooperativity between receptors. Our results suggest that the DMSO priming effect is due to potentiation of the Epo signaling pathway, thus resulting in a much more rapid and dramatic Epo-induced hemoglobinization response.