The tumor suppressor p53 is a sequence-specific transcription factor, which regulates the expression of target genes involved in different stress responses. To understand p53's essential transcriptional functions, unbiased analysis of its DNA-binding repertoire is pivotal. In a genome-wide tiling ChIP-on-chip approach, we have identified and characterized 1546 binding sites of p53 upon Actinomycin D treatment. Among those binding sites were known as well as novel p53 target sites, which included regulatory regions of potentially novel transcripts. Using this collection of genome-wide binding sites, a new high-confidence algorithm was developed, p53scan, to identify the p53 consensus-binding motif. Strikingly, this motif was present in the majority of all bound sequences with 83% of all binding sites containing the motif. In the surrounding sequences of the binding sites, several motifs for potential regulatory cobinders were identified. Finally, we show that the majority of the genome-wide p53 target sites can also be bound by overexpressed p63 and p73 in vivo , suggesting that they can possibly play an important role at p53 binding sites. This emphasizes the possible interplay of p53 and its family members in the context of target gene binding. Our study greatly expands the known, experimentally validated p53 binding site repertoire and serves as a valuable knowledgebase for future research.