Cells, infected with Friend virus, in culture afford a useful experimental system for studying the mechanisms of cell differentiation (1-4). In particular, the initiation of the specific differentiation program characteristic of erythropoiesis, including hemoglobin synthesis, may be studied with this cell system. Addition of dimethylsulfoxide to the culture medium induces erythroid differentiation (3). This paper examines the relationship of dimethylsulfoxide-induced hemoglobin synthesis in infected cells to the cell division cycle. Differentiation is generally accompanied by cell division (5). Whether prior or concurrent DNA synthesis is specifically required for the onset of transcription of those genes involved in a program of differentiation has not yet been determined. There are certain changes that occur during-S, G2, and M phases, but not in nondividing cells. These include the synthesis of DNA, and replacement and conformational rearrangement of regulatory proteins. It is possible that an agent that induces a transition in differentiation may act specifically during the dividing phase of a cycle of a cell. DNA synthesis and cell proliferation may be required for differentiation in a number of cell systems (5-14). Examples of a relationship between DNA synthesis and initiation of synthesis of proteins characteristic of the differentiated state come from studies on mammary tissue (6), and on uterus and oviduct (7), melanoma (8), erythropoietic (9-12), muscle (12, 13), myeloma (14), and lymphoid cells (15), as well as sporulation in Bacillus subtilis (16). These investigations, however, have not provided evidence whether the differentiative transition occurred during a specific phase of a particular cell cycle.In the present study, we attempt to determine if dimethylsulfoxide promotes expression of erythroid differentiation by an effect during a particular phase of the cell cycle. Using cells infected with Friend virus, strain 745A, synchronized by exposure to high levels of thymidine (17)