Small DNA viruses force quiescent cells to reenter the cell cycle in order to replicate their DNA. We report here that the adenovirus E1A protein creates an S phase environment in quiescent cells by overcoming the nucleosomal repression of E2F-targeted genes. These genes are surrounded by Lys-9-methylated H3 histones, and their promoters are occupied by the pRb-related protein p130 and the inhibitory transcription factor E2F4. Kinetic analysis indicates that E1A binds to E2F promoters where it eliminates p130 and E2F4, resulting in the dramatic elimination of H3 Lys-9 methylation. Thereafter, H3 Lys-9 acetylation occurs along with the recruitment of activating E2F family members, and this is followed by the transcriptional activity of E2F-targeted genes. These results indicate that E1A has a role in reconfiguring chromatin structure and that this activity is necessary to overcome the repressive mechanisms that maintain cells in a quiescent state.