The intra-S-phase checkpoint was among the first reported cell cycle checkpoints in mammalian cells. It transiently slows down the rate of DNA replication after DNA damage to facilitate repair and thus prevents genomic instability. The ionizing radiation (IR)-induced intra-S-phase checkpoint in mammalian cells is thought to be mainly dependent upon the kinase activity of ATM. Defects in the intra-S-phase checkpoint result in radio-resistant DNA synthesis (RDS), which promotes genomic instability. ATM belongs to the PI3K kinase family along with ATR and DNA-PKcs. ATR has been shown to be the key kinase for intra-S-phase checkpoint signaling in yeast and has also been implicated in this checkpoint in higher eukaryotes. Recently, contributions of DNA-PKcs to IR-induced G2-checkpoint could also be established. Whether and how ATR and DNA-PKcs are involved in the IR-induced intra-S-phase checkpoint in mammalian cells is incompletely characterized. Here, we investigated the contributions of ATM, ATR, and DNA-PKcs to intra-S-phase checkpoint activation after exposure to IR of human and hamster cells. The results suggest that the activities of both ATM and ATR are essential for efficient intra-S-phase checkpoint activation. Indeed, in a wild-type genetic background, ATR inhibition generates stronger checkpoint defects than ATM inhibition. Similar to G2 checkpoint, DNA-PKcs contributes to the recovery from the intra-S-phase checkpoint. DNA-PKcs–deficient cells show persistent, mainly ATR-dependent intra-S-phase checkpoints. A correlation between the degree of DSB end resection and the strength of the intra-S-phase checkpoint is observed, which again compares well to the G2 checkpoint response. We conclude that the organization of the intra-S-phase checkpoint has a similar mechanistic organization to that of the G2 checkpoint in cells irradiated in the G2 phase.