Previous studies have shown that exposure of cells to high levels of replicational stress leads to permanent proliferation arrest that does not require p53. We have examined cellular responses to therapeutically relevant low levels of replicational stress that allow limited proliferation. Chronic exposure to low concentrations of hydroxyurea, aphidicolin, or etoposide induced irreversible cell cycle arrest after several population doublings. Inhibition of p53 activity antagonized this arrest and enhanced the long-term proliferation of p53 mutant cells. p21CIP1 was found to be a critical p53 target for arrest induced by hydroxyurea or aphidicolin, but not etoposide, as judged by the ability of p21CIP1 suppression to mimic the effects of p53 disruption. Suppression of Rad51 expression, required for homologous recombination repair, blocked the ability of mutant p53 to antagonize arrest induced by etoposide, but not aphidicolin. Thus, the ability of mutant p53 to prevent arrest induced by replicational stress per se is primarily dependent on preventing p21CIP1 up-regulation. However, when replication stress is associated with DNA strand breaks (such as with etoposide), up-regulation of homologous recombination repair in response to p53 disruption becomes important. Since replicational stress leads to clonal selection of cells with p53 mutations, our results highlight the potential importance of chronic replicational stress in promoting cancer development.