The EWS/ETS fusion proteins associated with Ewings family tumors (EFTs) are thought to promote oncogenesis by acting as aberrant transcription factors. Uridine phosphorylase is a gene that is up-regulated by structurally distinct EWS/ETS fusions. Ectopic expression of uridine phosphorylase was able to support anchorage-independent cell growth, indicating that it plays an active role in the oncogenic process. Transcriptional up-regulation of uridine phosphorylase is shown to be mediated in a DNA binding-dependent manner, and reporter gene assays demonstrated that EWS/FLI1 and RAS mediate activation through a single activator protein 1/ETS site located in the uridine phosphorylase promoter. Chromatin immunoprecipitation assays reveal that EWS/FLI1 directly associates with the uridine phosphorylase promoter in vivo. Up-regulation of uridine phosphorylase by EWS/FLI1 sensitizes cells to growth inhibition by the pyrimidine analogue, 5′-deoxy-5′fluorouridine, both in tissue culture and in vivo model systems.