Overexpression of the E2F-1 cDNA in mammalian cells disrupts normal control of the cell cycle and drives cells into S phase. Whereas eliminating E2F activity would test its inferred involvement in the G~-S transition, elimination is complicated by the existence of gene families encoding mammalian E2F. Here we identify mutations in a single essential Drosophila gene, dE2F, that encodes a homolog of the mammalian E2F gene family. Embryos homozygous for null mutations of dE2F complete early cell cycles, presumably using maternal contributions of gene products, but DNA synthesis falls to virtually undetectable levels in cycle 17. Mutant embryos also lack the pulses of coordinate transcription of genes encoding replication functions that usually accompany each transition from quiescence to S phase. We conclude that in most cells clE2F is essential for a G~-S transcriptional program and for G~-S progression.[Key Words: Drosophila; E2F; S phase; cell cycle; embryogenesis] Received April 14, 1995; accepted May 3, 1995.The E2F transcription factor was originally identified through its role in the regulation of the adenovirus E2 promoter during viral infection. E2F is a heterodimer composed of a polypeptide encoded by DP-1 (or DP-1-related genes) and a polypeptide encoded by E2F-1 (or E2F-l-related genes). Studies in mammalian cells have suggested that E2F provides a nexus between the cyclindependent kinases, the retinoblastoma tumor suppressor gene, and the cell cycle control of transcription. A favored model of E2F regulation (for review, see Nevins 1992;Helin and Harlow 1993;Farnham 1995) is summarized below.In mammalian cells, the transcriptional activity of E2F is constrained by its physical association with the retinoblastoma protein (pRB) and the pRB-related proteins p107 and p130. The pRB-E2F interaction is regulated through phosphorylation by cyclin-dependent kinases (cdks). pRB has been shown to be phosphorylated by cdks that become activated during G1 phase of the cell cycle, such as cyclin D/cdk4, cyclin D/cdk6, and cyclin E/cdk2 kinases. Phosphorylation of pRB is thought to lead to the release of E2F, the activation of E2F-dependent transcription, and the promotion of entry into S phase. In this model, E2F and pRB are proposed to act antagonistically to regulate entry into S phase. This scheme provides an appealing common rationale for the action of oncogenes and tumor suppressor genes. Functions that interfere with the inhibitory interaction of 3Corresponding author.