Abstract Chk1 is a serine/threonine kinase that plays important roles in the cellular response to genotoxic stress. For this reason, there is a great deal of interest in using inhibitors of Chk1 to potentiate the effects of DNA-damaging chemotherapeutics. In addition, multiple studies have demonstrated that Chk1 activity is essential during an unperturbed cell cycle to ensure proper DNA replication and maintain genomic integrity. Therefore, it is plausible that a Chk1 inhibitor could also be efficacious as a single-agent therapeutic for human cancer. Here we show that treatment with Chk1-A, a potent and selective inhibitor of Chk1, alone is anti-proliferative against a wide array of cancer cell lines with varying degrees of potency. We sought to understand the mechanisms by which Chk1 inhibition derives the observed anti-proliferative effect. Employing the human leukemia cell line HEL92.1.7, a line particularly sensitive to Chk1 inhibition in terms of proliferation, we characterized the biochemical and functional effects of Chk1-A treatment. We observed concentration-dependent increases in phosphorylation of H2A. X, Chk1, and Chk2, which are markers of DNA damage and cell-cycle checkpoint activation. These biochemical events correlated with S-phase accumulation and eventual apoptosis. In vivo, we found that HEL92.1.7 tumor xenografts were sensitive to oral administration of Chk1-A at a dose that was well tolerated. Together, these studies suggest that inhibition of Chk1 results in DNA damage that induces apoptosis and that use of a Chk1 inhibitor as a single-agent could be an effective strategy to treat certain types of human cancers. Citation Format: {Authors}. {Abstract title} abstract. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3874.