Although recent work has suggested that lymphopenia-induced homeostatic proliferation may improve T cell-mediated tumor rejection, there is little direct evidence isolating homeostatic proliferation as an experimental variable, and the mechanism by which improved antitumor immunity occurs via homeostatic proliferation is poorly understood. An adoptive transfer model was developed in which tumor-specific 2C/RAG2(-/-) TCR transgenic CD8+ T cells were introduced either into the lymphopenic environment of RAG2(-/-) mice or into P14/RAG2(-/-) mice containing an irrelevant CD8+ TCR transgenic population. RAG2(-/-), but not P14/RAG2(-/-) recipients supported homeostatic proliferation of transferred T cells as well as tumor rejection. Despite absence of tumor rejection in P14/RAG2(-/-) recipients, 2C cells did become activated, as reflected by CFSE dilution and CD44 up-regulation. However, these cells showed poor IFN-gamma and IL-2 production upon restimulation, consistent with T cell anergy and similar to the hyporesponsiveness induced by administration of soluble peptide Ag. To determine whether homeostatic proliferation could uncouple T cell anergy, anergic 2C cells were transferred into RAG(-/-) recipients, which resulted in vigorous homeostatic proliferation, recovery of IL-2 production, and acquisition of the ability to reject tumors. Taken together, our data suggest that a major mechanism by which homeostatic proliferation supports tumor rejection is by maintaining and/or re-establishing T cell responsiveness.