Anti–CTLA-4 treatment improves the survival of patients with advanced-stage melanoma. However, although the anti–CTLA-4 antibody ipilimumab is now an approved treatment for patients with metastatic disease, it remains unknown by which mechanism it boosts tumor-specific T cell activity. In particular, it is unclear whether treatment amplifies previously induced T cell responses or whether it induces new tumor-specific T cell reactivities. Using a combination ultraviolet (UV)–induced peptide exchange and peptide–major histocompatibility complex (pMHC) combinatorial coding, we monitored immune reactivity against a panel of 145 melanoma-associated epitopes in a cohort of patients receiving anti–CTLA-4 treatment. Comparison of pre- and posttreatment T cell reactivities in peripheral blood mononuclear cell samples of 40 melanoma patients demonstrated that anti–CTLA-4 treatment induces a significant increase in the number of detectable melanoma-specific CD8 T cell responses (P = 0.0009). In striking contrast, the magnitude of both virus-specific and melanoma-specific T cell responses that were already detected before start of therapy remained unaltered by treatment (P = 0.74). The observation that anti–CTLA-4 treatment induces a significant number of newly detected T cell responses—but only infrequently boosts preexisting immune responses—provides strong evidence for anti–CTLA-4 therapy–enhanced T cell priming as a component of the clinical mode of action.