SummaryThe completion of the Arabidopsis genomic sequence offers the possibility to extract global information about regulatory mechanisms. Here, we describe a data mining strategy in combination with gene expression analysis to identify bona fide genes regulated by the E2F transcription factor. Starting with a genome‐wide search of chromosomal sites containing E2F‐binding sites, we studied in depth two of the most abundant E2F‐binding sites within the Arabidopsis genome and identified over 180 potential E2F target genes. Among them and in addition to cell cycle‐related genes, we have also identified genes belonging to other functional categories, e.g. transcription, stress and defense or signaling. We have determined the expression levels of genes selected from different categories under two experimental situations. Using cultured cells partially synchronized with aphidicolin, we found that most potential E2F targets identified in silico show a cell cycle‐regulated expression pattern with a peak in early/mid S‐phase. In addition, we used Arabidopsis transgenic plants expressing a DP gene containing a truncated DNA‐binding domain, which likely has a dominant‐negative effect on AtE2Fa, b and c (also named AtE2F3, 1 and 2, respectively), which require DP for efficient DNA binding. Contrary to the up‐regulation observed in early/mid S‐phase‐cultured cells, the expression of a large number of potential E2F targets was decreased in the transgenic plants. Our results strongly support that the RBR/E2F pathway plays a crucial role in regulating the expression of the genes identified in this study.