Previous studies have demonstrated cell cycle-dependent specificities in the interactions of E2F proteins with Rb family members. We now show that the formation of an E2F-p130 complex is unique to cells in a quiescent, G0 state. The E2F-p130 complex does not reform when cells reenter a proliferative state and cycle through G1. The presence of an E2F-p130 complex in quiescent cells coincides with the E2F-mediated repression of transcription of the E2F1 gene, and we show that the E2F sites in the E2F1 promoter are important as cells enter quiescence but play no apparent role in cycling cells. In addition, the decay of the E2F-p130 complex as cells reenter the cell cycle requires the action of G1 cyclin-dependent kinase activity. We conclude that the accumulation of the E2F-p130 complex in quiescent cells provides a negative control of certain key target genes and defines a functional distinction between these G0 cells and cells that exist transiently in G1.