The checkpoint sliding-clamp complex, Rad9/Rad1/Hus1, plays a critical role during initiation of checkpoint signals in response to DNA damage and replication disruption. We investigated the impact of loss of Rad1 on checkpoint function and on DNA replication in mammalian cells. We show that RAD1 is an essential gene for sustained cell proliferation and that loss of Rad1 causes destabilization of Rad9 and Hus1 and consequently disintegration of the sliding-clamp complex. In Rad1-depleted cells, Atr-dependent Chk1 activation was impaired whereas Atm-mediated Chk2 activation was unaffected, suggesting that the sliding clamp is required primarily in Atr-dependent signal activation. Disruption of sliding-clamp function also caused a major defect in S-phase control. Rad1-depleted cells exhibited an RDS phenotype, indicating that damage-induced S-phase arrest was compromised by Rad1 loss. Furthermore, lack of Rad1 also affected the efficiency of replication recovery from DNA synthesis blockage, resulting in a prolonged S phase. These deficiencies may perpetually generate DNA strand breakage as we have found chromosomal abnormalities in Rad1-depleted cells. We conclude that the Rad9/Rad1/Hus1 complex is essential for Atr-dependent checkpoint signaling, which may play critical roles in the facilitation of DNA replication and in the maintenance of genomic integrity.