The retinoblastoma susceptibility gene (Rb) participates in controlling the G1/S-phase transition, presumably by binding and inactivating E2F transcription activator family members. Mouse embryonic fibroblasts (MEFs) with no, one, or two inactivated Rb genes were used to determine the specific contributions of Rb protein to cell cycle progression and gene expression. MEFs lacking both Rb alleles (Rb-/-) entered S phase in the presence of the dihydrofolate reductase inhibitor methotrexate. Two E2F target genes, dihydrofolate reductase and thymidylate synthase, displayed elevated mRNA and protein levels in Rb- MEFs. Since absence of functional Rb protein in MEFs is sufficient for S-phase entry under growth-limiting conditions, these data indicate that the E2F complexes containing Rb protein, and not the Rb-related proteins p107 and p130, may be rate limiting for the G1/S transition. Antineoplastic drugs caused accumulation of p53 in the nuclei of both Rb+/+ and Rb-/- MEFs. While p53 induction led to apoptosis in Rb-/- MEFs, Rb+/- and Rb+/+ MEFs underwent cell cycle arrest without apoptosis. These results reveal that diverse growth signals work through Rb to regulate entry into S phase, and they indicate that absence of Rb protein produces a constitutive DNA replication signal capable of activating a p53-associated apoptotic response.