Several mammalian genes expressed in late G1 are positively regulated by E2F, a heterodimeric transcription factor. Genes encoding two E2F proteins, E2F-1 and DP-1, were regulated differently during the cell cycle and replicative senescence of normal human fibroblasts. In presenescent cells, E2F-1 mRNA was cell-cycle regulated, appearing a few hours before S phase. By contrast, DP-1 mRNA was constitutively expressed, independent of position in the cell cycle. After a finite number of divisions, normal cells enter a state of irreversible growth arrest termed senescence. Many genes remain mitogen-inducible in senescent cells; there are, however, exceptions, including several late G1 genes potentially regulated by E2F. Senescent cells expressed DP-1 at the presenescent level, but did not express E2F-1 mRNA. Senescent cells were also markedly deficient in E2F binding activity associated with the dihydrofolate reductase promoter. E2F-1 and DP-1 expression vectors only weakly induced DNA synthesis in quiescent or senescent human cells and immortal murine NIH3T3 cells, although the E2F-1 vector stimulated DNA synthesis in immortal murine A31 cells, and transactivated E2F-responsive promoters in NIH3T3 cells. The results suggest that senescent cells may fail to express late G1 genes due to repression of E2F-1, leading to a deficiency of E2F activity. Furthermore, although E2F-1 stimulates DNA synthesis in some cells, other cells, including normal human fibroblasts, require additional factors.