The invention of MHC-tetramer technology to label antigen-specific T cells has lead to a greatly enhanced understanding of T lymphocyte biology. This protocol describes the use of MHC class I tetramers to stain antigen-specific T cells in tissue sections. In situ tetramer staining (IST) can be used to determine the localization, abundance, and phenotype of antigen-specific T cells in native environments and in three-dimensional space. IST is broadly applicable because it can be used to stain essentially any antigen-specific T cell in any tissue for which MHC tetramers are available. IST can be combined with histochemistry and/or immunohistochemistry to permit visualization and characterization of antigen-specific T cells relative to other cell types in stained tissue sections. Thus, IST is a useful and valuable component of MHC-tetramer technology.