E2F is a family of transcription factors that regulates the cell cycle. It is widely accepted that E2F-mediated transactivation of a set of genes is the critical activity that governs cellular progression through G1 into S phase. In contrast to this hypothesis, we demonstrate that E2F actually suppresses the onset of S phase in two cell types when the cells are arrested by gamma irradiation. Our findings indicate that in these cells, the critical event triggering progression from G0/G1 arrest into S phase is the release of E2F-mediated transrepression of cell cycle genes, not transactivation by E2F. Furthermore, our data suggest that E2F-mediated transactivation is not necessary for the G1/S-phase transition in these cells.