Three sources of help for the development of a CD8+ CTL response have been described: the CD4+ direct and indirect pathways and the CD8+ direct pathway. In an effort to understand the minimal requirements for the development of a CTL response in vivo, we have bred mice transgenic for the 2C TCR onto a RAG(-/-) background. The 2C T cells in this animal are exclusively CD8+ CTLs of a single specificity, and they exhibit altered thymic maturation compared with that of T cells from 2C TCR/RAG(+/+) mice. T cells from 2C TCR/RAG(-/-) mice can be activated to a high level in vivo by administration of a self-MHC-restricted antigenic peptide. The 2C TCR/RAG(-/-) mice are able to reject B7-negative allogeneic tumors bearing the appropriate peptide/MHC ligand p2C/Ld. These mice fail to reject syngeneic tumors, and their RAG(-/-) littermates lacking 2C T cells uniformly succumb to both allogeneic and syngeneic tumors. Moreover, blockade of B7 costimulatory molecules fails to prevent tumor rejection in the 2C TCR/RAG(-/-) mice, suggesting that allorejection is occurring independently of B7-mediated costimulation as well as in the absence of CD4+ T cells. CTLs isolated from the site of the tumor during the period of rejection express the activation marker CD25 and are able to mediate ex vivo cytolysis of tumor cells bearing the appropriate Ag. These results suggest that in this TCR transgenic model with a very high precursor frequency, CTL development can occur in the absence of B7:CD28 costimulation and without CD4+ help.