CD4⁺ T cells selected by the type 1 diabetes associated class II MHC I-A^g7 molecules play a critical role in the disease process. Multivalent MHC/peptide tetramers have been used to directly detect antigen-specific T cells. Detection of autoantigen-activated CD4⁺ T cells with tetramers should be very helpful in the study of the roles of these cells in diabetes. We report here the generation of tetramers of I-A^g7 covalently linked to two glutamic acid decarboxylase (GAD) peptides and the detection of GAD peptide-activated T cells from nonobese diabetic (NOD) mice. The I-A^g7 heterodimers can form stable complexes with a covalently bound GAD peptide and can stimulate antigen specific T cells. Furthermore, I-A^g7/GAD peptide tetramer can detect most if not all of the antigen-specific CD4⁺ T cells from immunized NOD mice. Antigen-specific T cells detected by the tetramers can up-regulate their CD4 expression on the cell surface after being restimulated with the GAD peptides in vitro. In contrast, the tetramers can detect a percentage of T cells in lymph nodes and spleens and T cells infiltrating islets from nonimmunized mice that is not significantly above the background. Therefore, T cells specific for the GAD peptides are present in NOD mice at a frequency too low to be detected, but immunization of NOD mice can facilitate their detection by tetramers.