In an attempt to clarify the mechanisms of cytotoxicity mediated by CD4+ cytotoxic T lymphocytes (CTL), the expression of perforin and membrane-bound lymphotoxin (LT) (tumor necrosis factor-beta) in herpes simplex virus (HSV)-specific CD4+ human cytotoxic and noncytotoxic T- cell clones was examined. Three HSV-specific CD4+ human CTL clones that showed HLA-DR-restricted cytotoxicity and proliferative response were established. The cytotoxicity of these clones in 5-hour 51Cr release assays was found to be mediated by the directional target cell lysis and not by the release of cytotoxic soluble factors, ie, “innocent bystander” killing. Northern blot analysis showed that messenger RNAs for perforin and LT, which were both considered to be important mediators for cytotoxicity of CD8+ CTL and natural killer cells, were abundantly expressed in HSV-specific CD4+ CTL clones. Expression of perforin in the cytoplasm of CD4+ CTL clones was also detected by immunohistochemical staining using a monoclonal antibody against perforin. In addition, LT bound to the cell surface of CD4+ CTL clones was detected by flow cytometry. In contrast, little or no expression of perforin and LT was detected in three HSV-specific CD4+ noncytotoxic T- cell clones. Although the cytotoxicity mediated by lymphokine-activated killer cells was partly inhibited by addition of anti-LT antibody, it did not show any effect on the cytotoxicity of HSV-specific CD4+ CTL clones. In addition, it was found that cytotoxicity mediated by these CD4+ CTL clones was Ca2(+)-dependent. These data thus suggest that perforin and membrane-bound LT are both expressed in HSV-specific CD4+ CTL, although perforin might be the more important mediator in short- term culture.