The E2F pathway has been proposed to regulate genes involved in the transition from quiescence into DNA synthesis. However, this hypothesis has not been rigorously tested on a genomic scale. Toward this end, we have infected quiescent mouse fibroblasts, which do not express E2F1, with an E2F1-expressing adenovirus and examined the expression of more than 6000 genes using high-density microarrays. Microarray results clearly support the current paradigm; however, they suggest that E2F1 may also regulate unanticipated cellular functions including pathways involved in apoptosis, signal transduction, transcriptional control, and membrane biology. Most surprisingly, we identified a number of genes that are repressed by E2F1 expression, suggesting that E2F1 may have the potential to repress transcription of numerous genes through an unknown mechanism.