Rad9, a key component of genotoxin-activated checkpoint signaling pathways, associates with Hus1 and Rad1 in a heterotrimeric complex (the 9-1-1 complex). Rad9 is inducibly and constitutively phosphorylated. However, the role of Rad9 phosphorylation is unknown. Here we identified nine phosphorylation sites, all of which lie in the carboxyl-terminal 119-amino acid Rad9 tail and examined the role of phosphorylation in geno-toxin-triggered checkpoint activation. Rad9 mutants lacking a Ser-272 phosphorylation site, which is phosphorylated in response to genotoxins, had no effect on survival or checkpoint activation in Mrad9(-/-) mouse ES cells treated with hydroxyurea (HU), ionizing radiation (IR), or ultraviolet radiation (UV). In contrast, additional Rad9 tail phosphorylation sites were essential for Chk1 activation following HU, IR, and UV treatment. Consistent with a role for Chk1 in S-phase arrest, HU- and UV-induced S-phase arrest was abrogated in the Rad9 phosphorylation mutants. In contrast, however, Rad9 did not play a role in IR-induced S-phase arrest. Clonogenic assays revealed that cells expressing a Rad9 mutant lacking phosphorylation sites were as sensitive as Rad9(-/-) cells to UV and HU. Although Rad9 contributed to survival of IR-treated cells, the identified phosphorylation sites only minimally contributed to survival following IR treatment. Collectively, these results demonstrate that the Rad9 phospho-tail is a key participant in the Chk1 activation pathway and point to additional roles for Rad9 in cellular responses to IR.