Using an inducible transcription system which allows the regulated expression of C/EBP isoforms in tissue culture cells, we have found that the ectopic expression of C/EBP␣, at a level comparable to that found in normal liver tissue, has a pronounced antimitogenic effect in mouse L cells and NIH 3T3 cells. The inhibition of cell division by C/EBP␣ in mouse cells cannot be reversed by simian virus 40 T antigen, by oncogenic ras, or by adenovirus E1a protein. When expressed in thymidine kinase-deficient L cells or 3T3 cells, C/EBP␣ is detected in a protein complex which binds to the E2F binding sites found in the promoters of the genes for E2F-1 and dihydrofolate reductase (DHFR). Bacterially expressed C/EBP␣ has no affinity for these E2F sites, but when recombinant C/EBP␣ is added to nuclear extracts from mouse fibroblasts, a new E2F binding activity appears, which contains the C/EBP␣ protein. Using an E2F-DP1-responsive promoter linked to a reporter gene, it can be shown that C/EBP␣ directly inhibits the induction of this promoter by E2F-DP1 in transienttransfection assays. Furthermore, C/EBP␣ can be shown to inhibit the S-phase induction of the E2F and DHFR promoters in permanent cell lines. These findings delineate a straightforward mechanism for C/EBP␣-mediated cell growth arrest through repression of E2F-DP-mediated S-phase transcription.