The E2F family of transcription factors regulate genes, whose products are essential for progression through the mammalian cell cycle. The transcriptional activity of the E2Fs is inhibited through the specific binding of the retinoblastoma protein, pRB, and the pRB homologs p107 and p130 to their transactivation domains. Seven members of the E2F transcription factor family have been isolated so far, and we were interested in investigating the possible contribution of the various E2Fs to cell cycle control. By presenting the results of the generation of cell lines with tetracycline-controlled expression of E2F-1 and E2F-4 and microinjection of expression plasmids for all members of the E2F family, we demonstrate here that the pRB-associated E2Fs (E2F-1, E2F-2, and E2F-3) all induce S phase in quiescent rat fibroblasts when expressed alone. In contrast, the p107/p130-associated E2Fs require the coexpression of the heterodimeric partner DP-1 to promote S-phase entry and accelerate G 1 progression. Furthermore, the pRB-associated E2Fs were all able to overcome a G 1 arrest mediated by the p16 INK4 tumor suppressor protein, and E2F-1 was shown to override a G 1 block mediated by a neutralizing antibody to cyclin D1. The p16 INK4-induced G 1 arrest was not affected by expression of E2F-4, E2F-5, or DP-1 alone, but simultaneous expression of E2F-4 and DP-1 could overcome this block. Our results demonstrate that the generation of E2F activity is rate limiting for G 1 progression, is sufficient to induce S-phase entry, and overcomes a p16-mediated G 1 block, and since E2F-1, E2F-2, and E2F-3 are associated with pRB, they are the most likely downstream effectors in the p16-cyclin D-pRB pathway. Furthermore, our data suggest that the two subsets of E2Fs are regulated by distinct mechanisms and/or that they have distinct functions in cell cycle control. Since E2F-4 and E2F-5 cannot promote S-phase entry by themselves, our results may provide an explanation for the apparent lack of aberrations in p107 or p130 in human cancer.