Stalled replication forks induce p53, which is required to maintain the replication checkpoint. In contrast to the well-established mechanisms of DNA damage-activated p53, the downstream effectors and upstream regulators of p53 during replication blockade remain to be deciphered. Hydroxyurea triggered accumulation of p53 through an increase in protein stability. The requirement of p53 accumulation for the replication checkpoint was not due to p21(CIP1/WAF1) as its down-regulation with short-hairpin RNA did not affect the checkpoint. Similar to DNA damage, stalled replication triggered the activation of the MRN-ataxia telangiectasia mutated (ATM)/ATM and Rad3-related-CHK1/CHK2 axis. Down-regulation of CHK1 or CHK2, however, reduced p53 basal expression but not the hydroxyurea-dependent induction. Moreover, p53 was still stabilized in ataxia telangiectasia cells or in cells treated with caffeine, suggesting that ATM was not a critical determinant. These data also suggest that the functions of ATM, CHK1, and CHK2 in the replication checkpoint were not through the p53-p21(CIP1/WAF1) pathway. In contrast, induction of p53 by hydroxyurea was defective in cells lacking NBS1 and BLM. In this connection, the impaired replication checkpoint in several other genetic disorders has little correlation with the ability to stabilize p53. These data highlighted the different mechanisms involved in the stabilization of p53 after DNA damage and stalled replication forks.