Previous studies have demonstrated that herpes simplex virus-1 (HSV-1)-infected mononuclear cells are able to stimulate autologous peripheral blood mononuclear cells (PBMC) of immune donors and to activate HSV-specific cytotoxic T lymphocytes (CTL) expressing either gamma delta or alpha beta T-cell receptors (TCR). In the present report characterization of 10 gamma delta+ and six alpha beta+ HSV-specific cytotoxic T-cell colonies (TCC) is described. Cytotoxic colonies were derived from HSV-induced cell lines of three donors who, in previous experiments, had shown a prevalence of gamma delta+ or alpha beta+ effector cells. HSV-1 induced cell lines obtained from gamma delta responders included more than 80% of cells expressing V gamma 9/delta 2 TCR V region chains. gamma delta+ TCC also expressed V gamma 9/delta 2 molecules. alpha beta+ TCC all expressed CD8 antigen, while only one of 10 gamma delta+ TCC was CD8+, the others being CD4/CD8-double negative. The cytotoxic response of HSV-specific TCC was HLA-unrestricted; nevertheless CD8+ TCC were dependent on the expression of HLA class I on the surface of target cells to mediate cytolytic activity, while CD8- TCC were not. Blocking experiments with monoclonal antibody (mAb) specific for lymphocyte function-associated antigen-1 (LFA-1), which is expressed on all TCC, demonstrated that all alpha beta+ TCC and some gamma delta+ TCC also needed the interaction between LFA-1 and its ligands to develop cytotoxic activity. Altogether our data suggest that HSV-specific CTL may represent a population selected by a high concentration of antigen with a broad range of TCR affinities, which may play an important role as a first line of defence against HSV infection.