To determine the role of expanded CD4+ CD28 null T cells in multiple sclerosis and rheumatoid arthritis pathology, these cells were phenotypically characterized and their Ag reactivity was studied. FACS analysis confirmed that CD4+ CD28 null T cells are terminally differentiated effector memory cells. In addition, they express phenotypic markers that indicate their capacity to infiltrate into tissues and cause tissue damage. Whereas no reactivity to the candidate autoantigens myelin basic protein and collagen type II was observed within the CD4+ CD28 null T cell subset, CMV reactivity was prominent in four of four HC, four of four rheumatoid arthritis patients, and three of four multiple sclerosis patients. The level of the CMV-induced proliferative response was found to be related to the clonal diversity of the response. Interestingly, our results illustrate that CD4+ CD28 null T cells are not susceptible to the suppressive actions of CD4+ CD25+ regulatory T cells. In conclusion, this study provides several indications for a role of CD4+ CD28 null T cells in autoimmune pathology. CD4+ CD28 null T cells display pathogenic features, fill up immunological space, and are less susceptible to regulatory mechanisms. However, based on their low reactivity to the autoantigens tested in this study, CD4+ CD28 null T cells most likely do not play a direct autoaggressive role in autoimmune disease.