Studies in cell culture systems have indicated that oncogenic forms of Ras can affect apoptosis. Activating mutations of Ras occur in ~30% of all human tumors and 50% of colorectal carcinomas. Since these mutations appear at early or intermediate stages in multistep journeys to neoplasia, an effect on apoptosis may help determine whether initiated cells progress towards a more neoplastic state. We have tested the effects of K-ras(Val12) on apoptosis in transgenic mice. A lineage-specific promoter was used to direct expression of human K-ras(Val12), with or without wild-type (wt) or mutant SV-40 T antigens (TAg), in postmitotic villus enterocytes, the principal cell type of the small intestinal epithelium. Enterocytes can be induced to reenter the cell cycle by TAg(wt). Reentry is dependent upon the ability of TAg to bind pRB and is associated with a p53-independent apoptosis. Analyses of K-ras(Val12) x TAg(wt) bi-transgenic animals indicated that K-ras(Val12) can enhance this apoptosis threefold but only in cycling cells; increased apoptosis does not occur when K-ras(Val12) is expressed alone or with a TAg containing Glu107.108→Lys107.108 substitutions that block its ability to bind pRB. Analysis of bi-transgenic K-ras(Val12) x TAg(wt) mice homozygous for wild-type or null p53 alleles established that the enhancement of apoptosis occurs through a p53-independent mechanism, is not attributable to augmented proliferation or to an increase in abortive cell cycle reentry (compared to TAg(wt) mice), and is not associated with detectable changes in the crypt-villus patterns of expression of apoptotic regulators (Bcl-2, Bcl- X(L), Bak, and Bax) or mediators of epithelial cell-matrix interactions and survival (e.g., α5β1 integrin and its ligand, fibronectin). Coexpression of K-ras(val12) and TAg(wt) produces dysplasia. The K-ras(Val12)-augmented apoptosis is unrelated to this dysplasia; enhanced apoptosis is also observed in cycling nondysplastic enterocytes that produce K-ras(Val12) and a TAg with a COOH-terminal truncation. The dysplastic epithelium of K-ras(Val12) x TAg(wt) mice does not develop neoplasms. Our results are consistent with this finding: (a) When expressed in initiated enterocytes with a proliferative abnormality, K-ras(Val12)facilitates progression to a dysplastic phenotype; (b) by diminishing cell survival on the villus, the oncoprotein may impede further progression; and (c) additional mutations may be needed to suppress this proapoptotic response to K-ras(Val12).