Murine erythroleukemia cells, grown in culture and induced to differentiate by dimethylsulfoxide (DMSO), were employed to explore the relationship between cellular proliferation and maturation. The expression of an erythroid phenotype, as measured by the accumulation of hemoglobin, occurred over a narrow range of concentrations of inducers; the maximum degree of differentiation was attained at a level of inducing agent which caused slight inhibition of cell replication. Stationary phase cells with diminished capacity for DNA synthesis failed to differentiate in the presence of DMSO; whereas, in contrast, exponentially growing murine leukemia cells undergoing extensive DNA biosynthesis readily attained a differentiated phenotype. The induced synthesis of hemoglobin in Friend cells exposed to DMSO was inhibited by 5-bromo-2'-deoxyuridine, arabinosylcytosine and hydroxyurea, when cells were simultaneously exposed to DMSO and a metabolic inhibitor. However, addition of either 5-bromo-2'-deoxyuridine, arabinosylcytosine or hydroxyurea to cultures pretreated with DMSO did not prevent the ultimate expression of the erythroid phenotype. These findings suggest that a process which is associated with cellular proliferation is required for the initiation of murine erythroleukemia cell maturation, but not for the ultimate accumulation of the erythroid marker hemoglobin in cells programmed to differentiate.