Application of tetrameric MHC class I-peptide complexes has significantly improved the monitoring of antigen-specific T cell immune responses in mouse models as well as in clinical studies. Especially MHC class I tetramer analysis of tumor-specific T cells in suspension or on thick vibratome sections from viable tissue has been proven extremely useful. Using the well-characterized mouse tyrosinase-related-protein-2 specific cytotoxic T cell (CTL) clone LP9, we now developed a method that allows for specific identification of T cells with MHC class I tetramers in 8 mum thick, chemically fixed cryosections. The protocol was validated in a murine influenza virus-infection model. Moreover, analysis of delayed type hypersensitivity (DTH) skin biopsies from melanoma patients vaccinated with peptide-loaded mature dendritic cells, revealed the presence and location of anti-tumor CTLs. The specificity of the CTLs detected in situ correlated with both the DTH challenge specificity and reactivity of cell suspensions derived from the same biopsies. Collectively, our data demonstrate that in situ MHC class I tetramer staining provides a valuable tool to reveal the presence and anatomical location of specific CTLs in frozen tissue following immune-based treatment strategies in cancer patients.