Virus-specific cytotoxic T lymphocytes (CTLs) have been suggested to be responsible for the liver injuries in patients with hepatitis C virus (HCV) infection. However, there has been no report of direct evidence to substantiate this hypothesis. In this study, we performed in vitro autologous hepatocytotoxicity assay in 45 patients to examine a possible role of CTLs to HCV-in-fected liver cells. The data were correlated with histology activity index of liver biopsy specimens. Lymphocyte subsets and hepatocyte expression of human major histocompatibility complex antigens class I and class II (HLA-I and HLA-II) were also evaluated. The immunohistochemical study showed more prominent HLA-I expression than HLA-II on hepatocytes (mean score ± SEM: 2.34 ± 0.11 vs. 0.42 ± 0.08; P < .01). The lymphocyte subset analysis showed that CD8+ T cells were dominant in the lobular areas showing spotty necrosis, whereas CD4+ T cells were prominent in the portal and periportal areas (P < .01). Most patients had a significant T cell-mediated cytotoxicity to hepatocytes as compared with non-T cells (percentage cytotoxicity ± SEM: 46.4 ± 2.3 vs. 13.8 ± 2.7; P < .001). T cell-mediated hepatocytotoxicity had a linear correlation with HAI (P < .05). The T cell-mediated cytotoxicity could be blocked by anti-CD8 (43.7% vs. 9.5%, P < .05) or by anti-HLA-I (43.7% vs. 18.5%, P < .05) but not by anti-CD4 or anti-HLA-II monoclonal antibodies. These findings strongly suggest that HLA-I–restricted, CD8+ T cell–mediated hepatocytotoxicity is an important pathogenetic mechanism in patients with chronic HCV infection. (Hepatology 1995; 22:1368–1373).