Autoreactive T cells have recently been detected not only in autoimmune diseases but also in healthy individuals, but their frequency is thought to be low. The aim of our study was to estimate the frequency of self-reactive T cells by using limiting dilution analyses of peripheral blood lymphocytes. Assessment of self-reactivity in this study was defined as T-cell proliferation to autologous non-T cells in the absence of foreign antigens. When culture conditions were optimized by adding interleukin 2, healthy individuals showed a frequency of self-reactive T cells ranging from 1/60 to 1/600. These results were confirmed by using unseparated peripheral blood leukocytes or Epstein-Barr virus transformed B-cell blasts as stimulators. All cultures were performed exclusively in autologous serum. Single cell cloning from a healthy donor yielded 568 T-cell clones, 12 of which showed self-reactivity giving a frequency of more than 1 in 50 T cells. Eight of these 12 T-cell clones were inhibited by MHC-class II antibodies. Frequency analyses of self-reactive T cells in patients with autoimmune diseases (rheumatoid arthritis, autoimmune hepatitis or primary biliary cirrhosis), with viral hepatitis or with inflammatory bowel diseases showed similar frequencies in all patient groups and no significant differences from normal individuals. In conclusion, we have found a high frequency of self-reactive T cells in both health and disease. We postulate that self-reactive T cells constitute an important part of the physiological T-cell repertoire.