Changes occur in polyamine metabolism which are associated with differentiation of Friend erythroleukemia cells in culture. The intracellular distribution of the polyamines changes, the absolute level of each of the major polyamine falls, and the uptake and metabolism of exogenous putrescine is altered during the course of differentiation. We have examined the labeling of putrescine metabolites after the administration of radioactive putrescine, either as a single dose added at the onset of induction, or as a pulse label added at various times during the course of differentiation. In these experiments we measured both acid-soluble and acid-precipitable radioactivity. The conversion of putrescine to gamma-aminobutyric acid is enhanced in the cell induced to differentiate, and the acid-precipitable fraction rises as the percentage of total cell-associated radioactivity in the induced cell. We have identified hypusine [N-epsilon-(4-amino-2-hydroxybutyl)lysine] as a major radioactive component of a single radioactive Mr 18,000 protein band in both the noninduced and the induced cells.