Friend erythroleukemia cells grown in culture and induced to differentiate along the erythroid developmental pathway by dimethyl sulfoxide (DMSO) were used as a model system to investigate the requirement for cellular replication to express a differentiated erythroid phenotype. That cytokinesis is not essential for DMSO-induced erythroid differentiation as measured by the synthesis and accumulation of hemoglobin was shown by experiments using cytochalasin B. In these studies, hemoglobin was found to accumulate in Friend cells treated simultaneously with DMSO and cytochalasin B; such treatment caused cells to become enlarged and multinucleated due to inhibition of cytokinesis by cytochalasin B. In contrast, exposure of cells to cytochalasin B for at least 48 hr prior to DMSO caused significant inhibition of erythroid differentiation. The findings support the concept that cellular division and, thereby the production of new cellular types are not required for gene activation and the expression of an erythroid phenotype. These effects of cytochalasin B on DMSO-induced differentiation of Friend leukemia cells also suggest plasma membrane-cytoskeleton involvement in the initiation of the erythroid maturation process in this system.