The low precursor frequency of Ag-reactive CD4+ T cells has been a barrier to the study of CD4+ T cell responses to conventional Ags as well as CD4+ T cell responses to autoantigens recognized during the course of an autoimmune disease. We have recently reported that all “conventional Ag” reactive CD4+ T cells are contained within the subpopulation expressing high levels of the CD4 molecule, termed CD4 high. We have identified a CD4 high population in the islets of Langerhans of prediabetic nonobese diabetic (NOD) mice that is extremely potent in transferring disease. As few as 500 CD4 high islet-infiltrating CD4+ T cells transferred insulin-dependent diabetes mellitus to CD8 reconstituted NOD-SCID mice within 30 days of transfer. In contrast, CD4 high T cells isolated from either NOD spleen or salivary glands did not transfer insulin-dependent diabetes mellitus into similar CD8-reconstituted NOD-SCID recipients. These data indicate that the precursor frequency of NOD islet-reactive, pathogenic CD4+ T cells is much higher in the prediabetic NOD pancreas than in these other organs. The islet-infiltrating CD4 high T cells displayed selected memory markers, by cell surface analysis, and displayed a Th 1 phenotype by RNase protection assay, but had a marked decrease in IL-4 mRNA determined by quantitative real time PCR when compared with the less pathogenic CD4 normal islet-infiltrating T cells. Use of the CD4 high marker to select Ag activated T cells represents a tool to isolate and study pathogenic CD4+ T cells from autoimmune lesions in which the Ag has not been previously defined.