Tracking antigen specific T cells with major histocompatibility complex (MHC) tetramers has provided us with insights into the dynamics of the adaptive immune system and holds great promise to aid in patient management and drug and vaccine development. Progress has been made primarily using MHC class I tetramers to monitor CD8(+) T cells, whereas corresponding efforts to stain CD4(+) T cells with class II tetramers have not been as successful. Two major reasons have been proposed for this lack of progress: (1). The frequency of antigen-specific CD4(+) T cells is lower than the frequency of CD8(+) T cells and (2). some, but not all, antigen- specific CD4(+) T cells can bind tetramer because of low functional avidity. In this study, we asked if CD4(+) T cells specific for common human viruses (e.g., influenza and Epstein-Barr) can be detected in healthy individuals previously exposed to them. We were able to clearly detect specific CD4(+) T cells in all donors after in vitro expansion of peripheral blood mononuclear cells. Furthermore, we observe a clear separation of tetramer negative and tetramer positive CD4(+) T cells in most samples similar to patterns commonly seen with class I tetramers. The data indicate that MHC class II tetramers can be used reliably for the identification of CD4(+) T cells specific for ubiquitous infectious agents in normal donors.