Cell-cycle withdrawal is the irreversible arrest of replication that occurs in keratinocytes early in terminal differentiation. According to the epidermal proliferation unit (EPU) model of renewal, withdrawal takes place in a subset of cells that have completed a final cycle of amplification replication. Using a recently developed double-labelling assay, we followed cell-cycle withdrawal in growing cultures of epidermal keratinocytes and correlated these results with population-growth kinetics. The levels of withdrawal measured were much too high to be consistent with the population-growth kinetics. These unexpectedly high levels could be explained by postulating that withdrawal takes place in a specific subset of cells, as described by the EPU model. Other possible explanations were entertained but, for various reasons, were considered unlikely. To learn whether withdrawal occurred in basal or suprabasal cells, confluent cultures were pulse labeled with 3H-thymidine, and the position of the labeled cells was monitored by autoradiography during the period of cold chase. The results indicated that, in cultured keratinocytes, withdrawal takes place while the cells are still in the basal compartment.