The E2F1 transcription factor is a critical regulator of cell cycle due to its ability to promote S phase entry. However, E2F1 overexpression also sensitizes cells to apoptosis and E2F1-null mice are predisposed to tumor development, suggesting that it also has properties of a growth suppressor. E2F1 transcription function is regulated by interaction with hypophosphorylated pRb. Cdk inhibitors such as p16INK4a and p27Kip1 inhibit pRb phosphorylation by the cyclin D/Cdk4 and cyclin E/Cdk2 complexes, thus keeping E2F1 in an inactive state. We found that E2F1 binds to the p27 promoter in vivo and activates p27 mRNA and protein expression. Depletion of endogenous E2F1 by siRNA causes a reduction in basal p27 expression level. Inhibition of endogenous p27 expression by siRNA increases E2F1 transcriptional activity and permits accelerated cell cycle progression by exogenous E2F1. These observations suggest that induction of p27 acts as a negative feedback mechanism for E2F1 and may also contribute to other functions of E2F1.