We established CTL lines and clones against colonic epithelial cells from PBLs of patients with ulcerative colitis by continuous stimulation with HLA-A locus-matched colonic epithelial cell lines. We developed a nonradioactive europium release cytotoxicity assay to detect CTLs. PBLs from 3 of 12 patients but not from any of 14 normal controls who shared at least one haplotype of HLA-A locus with two colonic epithelial cell lines, CW2 and ACM, showed increased cytotoxicity against these lines. Three CTL lines established from the PBLs of patients showed increased cytotoxicity against HLA-A locus-matched CW2 or ACM but not against matched lung or esophagus cell lines. The phenotypes of CTL lines were alpha beta-TCR+ CD3+ CD8+ CD16-. The CTL line MS showed increased cytotoxicity against freshly isolated colonic epithelial cells but not against cells with a different HLA-A locus. Two CTL clones were generated from MS and clone 3-2, expressing CD3+ CD8+ CD4- CD56-, showed high MHC class I-restricted cytotoxicity against the colonic epithelial cells. These results indicated that CTLs against colonic epithelial cells may contribute to epithelial cell damage in ulcerative colitis.