The E2F transcription factors are essential regulators of cell growth in multicellular organisms, controlling the expression of a number of genes whose products are involved in DNA replication and cell proliferation. In Saccharomyces cerevisiae, the MBF and SBF transcription complexes have functions similar to those of E2F proteins in higher eukaryotes, by regulating the timed expression of genes implicated in cell cycle progression and DNA synthesis. The CDC6 gene is a target for MBF and SBF-regulated transcription. S. cerevisiae Cdc6p induces the formation of the prereplication complex and is essential for initiation of DNA replication. Interestingly, the Cdc6p homolog in Schizosaccharomyces pombe, Cdc18p, is regulated by DSC1, the S. pombe homolog of MBF. By cloning the promoter for the human homolog of Cdc6p and Cdc18p, we demonstrate here that the cell cycle-regulated transcription of this gene is dependent on E2F. In vivo footprinting data demonstrate that the identified E2F sites are occupied in resting cells and in exponentially growing cells, suggesting that E2F is responsible for downregulating the promoter in early phases of the cell cycle and the subsequent upregulation when cells enter S phase. Our data also demonstrate that the human CDC6 protein (hCDC6) is essential and limiting for DNA synthesis, since microinjection of an anti-CDC6 rabbit antiserum blocks DNA synthesis and CDC6 cooperates with cyclin E to induce entry into S phase in cotransfection experiments. Furthermore, E2F is sufficient to induce expression of the endogenous CDC6 gene even in the absence of de novo protein synthesis. In conclusion, our results provide a direct link between regulated progression through G 1 controlled by the pRB pathway and the expression of proteins essential for the initiation of DNA replication.Although E2F was originally defined as a factor that binds specifically to an element in the adenovirus E2 promoter (42), it is now evident that E2F is essential for coordinating transcription during the mammalian cell cycle (for reviews, see references 12 and 72). A number of genes are found to be regulated by E2F, particularly during the transition from G 1 to S phase. To date, six members of the E2F family are known: E2F-1 through E2F-5 and the recently identified E2F-6 (11). Furthermore, two heterodimerization partners of the E2Fs, DP-1 and DP-2, have been isolated. The E2F transcription factors appear to be key downstream targets for the retinoblastoma protein pRB and two pRB-related proteins, p107 and p130 (reviewed in references 12 and 70). Binding of pRB family members (also called pocket proteins) to the E2F transcription factors results in transcriptional repression of E2F-regulated genes. Phosphorylation of the pocket proteins by cyclin-dependent kinases releases the pocket proteins from E2F, leading to derepression and/or activation of E2F-dependent genes and subsequent entry into S phase. The demonstration that deregulated E2F activity is sufficient to induce S phase in quiescent cells has provided a m...