CD4+ T cells are thought to contribute to antiviral immune responses by secretion of cytokines thereby providing help to CD8+ T and B cells. However, perforin-positive cytotoxic CD4+ T cells have been described in human immunodeficiency virus-positive patients suggesting a role not only of CD8+ but also of CD4+ T cells for killing virus-infected cells. We investigated 76 patients with viral hepatitis [15 hepatitis B virus (HBV), 22 HBV/hepatitis D virus and 17 hepatitis C virus (HCV)] for cytotoxic CD4+ T cells. The frequency of perforin-positive CD4+ T cells in viral hepatitis was highly variable ranging from < 1% to more than 25%. Perforin-positive CD4+ T cells displayed the phenotype of terminally differentiated effector cells (CD28-, CD27-). The highest frequencies of CD4+ cytotoxic T lymphocytes (CTLs) were found in patients with delta hepatitis (P = 0.04 vs HBV and HCV patients), and the presence of CD4+ CTLs was associated with elevated aspartate aminotransferase levels (P = 0.01) and decreased platelet counts (P = 0.03). Perforin-positive CD4+ T cells decreased in two individuals during spontaneous clearance of acute hepatitis C. Significant associations were found between the frequency of perforin-expressing CD4+ cells and age (P = 0.04), perforin-positive CD8+ cells (P < 0.001) and perforin-positive CD4-/CD8- lymphoid cells (P = 0.002). Differentiated CD27- effector CD4+ CTLs can be detected in patients with viral hepatitis. In particular in patients with more advanced liver disease, the accumulation of perforin-positive T cells with age could be one correlate for the more severe course of viral hepatitis in elderly individuals.