E2F/DP heterodimers play a pivotal role in the regulation of cell growth and differentiation. A decrease in E2F/DP activity occurs during cell cycle arrest and differentiation. However, very little is known about the specific role of the various E2F/DP members along the transition from proliferation to terminal differentiation. We have previously shown that E2F4 accounts for the vast majority of the endogenous E2F in differentiating muscle cells. Here, we show that E2F4, which lacks a nuclear localization signal (nls), is distributed in both the nucleus and the cytoplasm, in either asynchronously growing myoblasts or differentiated myotubes. E2F4 nuclear accumulation is induced by the binding in the cytoplasm with specific partners p107, pRb2/p130, and DP3delta, an nls-containing spliced form of DP3, which provide the nls. Although overexpression of E2F4/DP3delta reactivates the cell cycle in quiescent cells, the E2F4 nuclear accumulation induced by pRb2/p130 and p107 correlates with cell growth arrest Moreover, E2F4/DP3delta-induced cell cycle reactivation is efficiently counteracted by either p107 or pRb2/p130 overexpression. Reinduction in quiescent cells of DNA synthesis by E2F1/DP1 overexpression is abrogated by coexpression of pRb and is hampered by MyoD overexpression. Both pRb2/p130 and pRb, as well as MyoD, are up-regulated in myotubes. Accordingly, multinucleated myotubes, which are induced to reenter the S-phase by oncoviral proteins, are refractory to cell cycle reactivation by forced expression of E2F4/DP3delta or E2F1/DP1. Thus, E2F/DP repression represents only one of multiple redundant circuits that control the postmitotic state in terminally differentiated cells and that are targeted by adenovirus E1A and SV40 large T antigen.