The transcription factor E2F mediates cell cycle-dependent expression of genes important for cell proliferation in response to growth stimulation. To further understand the role of E2F, we utilized a sensitive subtraction method to explore new E2F1 targets, which are expressed at low levels and might have been unrecognized in previous studies. We identified 33 new E2F1-inducible genes, including checkpoint genes Claspin and Rad51ap1, and four genes with unknown function required for cell cycle progression. Moreover, we found three groups of E2F1-inducible genes that were not induced by growth stimulation. At least, two groups of genes were directly induced by E2F1, indicating that E2F1 can regulate expression of genes not induced during the cell cycle. One included Neogenin, WASF1 and SGEF genes, which may have a role in differentiation or development. The other was the cyclin-dependent kinase inhibitor p27(Kip1), which was involved in suppression of inappropriate cell cycle progression induced by deregulated E2F. E2F1-responsive regions of these genes were located more upstream than those of typical E2F targets and did not have typical E2F sites. These results indicate that there are groups of E2F1 targets, which are regulated in a distinct manner from that of typical E2F targets.