A new technique that monitors cell cycle progression over multiple cycles was used to gain insight into how p53 limits the emergence of variants with structural chromosome alterations following gamma-irradiation. G0-synchronized, p53+ (with a functional p53 pathway) normal human fibroblast and epithelial strains underwent a dose-dependent permanent arrest in the initial G0-G1 phase after irradiation. The dose-response curves indicate that a single event, such as an irreparable DNA break, may be sufficient to induce arrest. p53+ cells that escaped the initial G0-G1 phase after irradiation entered S phase in at least two waves. However, many of these cells underwent long-term arrest in subsequent phases. In contrast, virtually all of the cells in isogenic p53- (with a nonfunctional p53 pathway) strains escaped from the first G0-G1 phase without delay, regardless of the dose. p53- cells were also eliminated in subsequent phases but at significantly lower frequencies. Consistent with these findings, the reproductive viability of p53- cells was higher than p53+ cells. The nonclonogenic fraction appeared to be eliminated within three cycles for both cell types. In addition, artificial holding in G0 after irradiation, which allows for the repair of potentially lethal damage, led to similar increases in survival in p53+ and p53- cells. These data are inconsistent with the hypothesis that the primary function of p53-dependent G0-G1 arrest in response to gamma-irradiation is to allow additional time for DNA repair. Rather, they indicate that p53 helps maintain genetic stability by eliminating cells with damaged chromosomes from the reproductively viable population.