Premature chromatin condensation (PCC) is a hallmark of mammalian cells that begin mitosis before completing DNA replication. This lethal event is prevented by a highly conserved checkpoint involving an unknown, caffeine-sensitive mediator. Here, we have examined the possible involvement of the caffeine-sensitive ATM and ATR protein kinases in this checkpoint. We show that caffeine’s ability to inhibit ATR (but not ATM) causes PCC, that ATR (but not ATM) prevents PCC, and that ATR prevents PCC via Chk-1 regulation. Moreover, mimicking cancer cell phenotypes by disrupting normal G(1) checkpoints sensitizes cells to PCC by ATR inhibition plus low-dose DNA damage. Notably, loss of p53 function potently sensitizes cells to PCC caused by ATR inhibition by a small molecule. We present a molecular model for how ATR prevents PCC and suggest that ATR represents an attractive therapeutic target for selectively killing cancer cells by premature chromatin condensation.