The highly specific interaction between major histocompatibility complex (MHC)/peptide and its cognate T-cell receptor (TCR) directs to the specificity of the T-cell response. Although the interaction affinity of individual MHC/peptide and TCR molecules is low, multimerization of the MHC/peptide complexes increases the avidity of their interaction with TCRs, and enables the complexes to be used as staining reagents for antigen-specific T cells. Different approaches are used to generate class I and class II tetramer reagents, and these reagents have been demonstrated to be useful in identifying CD8+ and CD4+ T cells, respectively. This article focuses on the uses of human lymphocyte antigen (HLA) class II tetramers in tracking antigen-specific T cells. Class II tetramers can also be used to map antigenic epitopes of known antigens through a novel peptide screening procedure known as tetramer-guided epitope mapping (TGEM). In this article, we provide a description of the methodological approach of using HLA class II tetramers in tracking and isolating T cells, and the use of class II tetramers in mapping T-cell epitopes.