Abstract Immune-mediated tumor rejection relies on fully functional T-cell responses and neutralization of an adverse tumor microenvironment. In clinical trials, we detected peptide-specific but non–tumor-reactive and therefore not fully functional CD8+ T cells post-vaccination against tumor antigens. Understanding the molecular mechanisms behind nontumor reactivity will be a prerequisite to overcome this CD8+ T-cell deviation. We report that these non–tumor-reactive CD8+ T cells are characterized by a molecular program associated with hallmarks of “division arrest anergy.” Non–tumor-reactive CD8+ T cells are characterized by coexpression of CD7, CD25, and CD69 as well as elevated levels of lckp505 and p27kip1. In vivo quantification revealed high prevalence of non–tumor-reactive CD8+ T cells with increased levels during cancer vaccination. Furthermore, their presence was associated with a trend toward shorter survival. Dynamics and frequencies of non–target-reactive CD8+ T cells need to be further addressed in context of therapeutic vaccine development in cancer, chronic infections, and autoimmune diseases. [Cancer Res 2009;69(10):4346–54]