Cell cycle checkpoints are biochemical signal transduction pathways that prevent downstream events from being initiated until upstream processes are completed. We analyzed whether the p53 or pRb tumor suppressors are involved in a checkpoint(s) that prevents DNA rereplication in the presence of drugs that interfere with spindle assembly. Normal mouse and human fibroblasts arrested with a 4N DNA content when treated with nocodazole and Colcemid, whereas isogeneic p53-deficient or pRb-deficient derivatives became polyploid. Flow cytometric and cytogenetic analyses demonstrated that the polyploidy resulted from genome-wide rereplication without an intervening mitosis. Thus, p53 and pRb help maintain normal cell ploidy by preventing DNA rereplication prior to mitotic division.