ARTICLE ABSTRACTIneffective recognition of tumor cells by CD8+ T cells is a limitation of cancer immunotherapy. Therefore, treatment regimens that coordinately promote enhanced antitumor CD8+ T-cell activation, delivery, and target cell recognition should yield greater clinical benefit. Using an MCA205 sarcoma model, we show that in vitro treatment of tumor cells with the HSP90 inhibitor 17-DMAG results in the transient (proteasome-dependent) degradation of the HSP90 client protein EphA2 and the subsequent increased recognition of tumor cells by Type-1 anti-EphA2 CD8+ T cells. In vivo administration of 17-DMAG to tumor-bearing mice led to slowed tumor growth, enhanced/prolonged recognition of tumor cells by anti-EphA2 CD8+ T cells, reduced levels of myeloid-derived suppressor cells and regulatory T cells in the tumor microenvironment, and activation of tumor-associated vascular endothelial cells in association with elevated levels of Type-1 tumor-infiltrating lymphocytes. When combined with EphA2-specific active vaccination or the adoptive transfer of EphA2-specific CD8+ T cells, 17-DMAG cotreatment yielded a superior tumor therapeutic regimen that was capable of rendering animals free of disease. Taken together, our findings indicate that 17-DMAG functions as an immune adjuvant in the context of vaccines targeting EphA2. Cancer Res; 72(13); 3196–206. ©2012 AACR.