The tumor suppressor p53 plays an essential role in cellular adaptation to stress. In response to ionizing radiation, p53 regulates the transcription of genes in a diverse set of pathways including DNA repair, cell cycle arrest, and apoptosis. Previously, we identified by microarray analysis a set of genes that are transcriptionally activated or repressed in response to radiation exposure. In this study, we use computational methods and molecular techniques, including location analysis (ChIP-on-chip assay), to identify ionizing radiation-responsive genes that are directly regulated by p53. Among the 489 ionizing radiation-responsive genes examined, 38 genes were found to be p53 targets. Some of these genes are previously known to be directly regulated by p53 whereas others are novel p53 targets. We further showed that the novel p53 target genes are transcriptionally regulated by p53. The binding of p53 to promoters of target genes correlated with increased transcript levels of these genes in cells with functional p53. However, p53 binding and subsequent transcriptional activation of these target genes were significantly diminished in cells with mutant p53 and in cells from patients with ataxia telangiectasia, which have impaired p53 activation following ionizing radiation exposure. Identification and characterization of ionizing radiation-responsive p53 target genes extend our knowledge of the diverse role that p53 plays in the DNA damage response.