Abstract Eya proteins are transcriptional co-activators of the homeobox gene Six1 and are also unique protein tyrosine phosphatases. Six1 and Eya2 are required for normal development but are down-regulated in most adult tissues. However, Six1 and Eya are over-expressed in a large number of breast tumors and play a causal role in the initiation and development of these tumors. The phosphatase activity of Eya was shown to be important for the transformation, migration, invasion, and metastasis of breast cancer cells. Eya's phosphatase activity therefore provides an attractive enzymatic target for anti-breast cancer therapy. In addition, Eyas also direct cells to a DNA-damage-repair pathway instead of an apoptotic pathway upon DNA damage through the dephosphorylation of histone H2AX. Inhibiting Eya phosphatase activity can therefore potentially sensitize tumor tissue to radiation or chemotherapy treatment. We developed an HTS assay to identify small molecule inhibitors of Eya's phosphatase activity. In collaboration with the NIH Chemical Genomics Center, we have screened over 300,000 compounds and identified several promising lead compounds. These hits are currently being evaluated for their potency, specificity, and mechanism of action using biochemical, structural, and cell-based assays. We believe that Eya is a significant target for cancer therapy through sensitizing cancer cells to radiation and chemotherapy and through the inhibition of Six1-mediated tumorigenesis and metastasis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1378. doi:10.1158/1538-7445.AM2011-1378