To analyze the nature of intestinal intraepithelial lymphocytes (iIELs) in inflammatory intestinal disease, we established T cell lines of iIELs isolated from endoscopic biopsied ileal and colonic mucosa of Crohn's disease patients. Seven T cell lines from the inflamed terminal ileum of 13 patients, but none of 16 T cell lines from normal terminal ileum, have shown a deviation of T cell receptor variable region gene usage and were enriched in the proportion of CD4+V beta 5.2/5.3+ cells. CD4+V beta 5.2/5.3+ cells in the T cell lines were not increased after stimulation with purified protein derivatives or 65-kDa heat-shock protein but significantly increased after stimulation with staphylococcal enterotoxins C1 and D. Those cells showed increased cytolytic activity against target cells cross-linked by anti-V beta 5.2/5.3 and produced a large amount of interferon-gamma. These results indicated that CD4+V beta 5.2/5.3+ iIELs were preferentially activated in the inflamed lesions of Crohn's disease and may play a possible role in the triggering and progression of human inflammatory intestinal disease.