The integrity of the human genome is preserved by signal transduction pathways called checkpoints, which delay progression through the cell cycle when DNA damage is present. Three checkpoint proteins, hRad9, hRad1, and hHus1, form a proliferating cell nuclear antigen-like, heterotrimeric complex that has been proposed to function in the initial detection of DNA structural abnormalities. hRad9 is highly modified by phosphorylation, in a constitutive manner and in response to both DNA damage and cell cycle position. Here we present evidence that Thr292 of hRad9 is subject to Cdc2-dependent phosphorylation in mitosis. Furthermore, our data are also consistent with four other hRad9 phosphorylation sites (Ser277, Ser328, Ser336, and Thr355) being regulated in part by Cdc2. We also identify Ser387 as a novel site of hRad9 constitutive phosphorylation and show that phosphorylation at Ser387 is a prerequisite for one form of DNA damage-induced hyperphosphorylation of hRad9. Characterization of nonphosphorylatable mutants has revealed that hRad9 phosphorylation plays a critical role in checkpoint signaling. Overexpression of these mutants blocks the interaction between hRad9 and the DNA damage-responsive protein TopBP1 and impairs the cellular response to DNA damage during S phase.