The characterization of HSV-specific human CTL, obtained in short term cultures by stimulating PBMC of healthy HSV-immune donors with autologous, PHA-activated, HSV-1-infected mononuclear cells, is described. CTL induced by using this technique are able to mediate a strong lytic activity against both HSV-1- and HSV-2-infected targets, whereas they do not kill autologous EBV-lymphoblastoid cell lines unless they are superinfected with HSV-1. TCR-gamma/delta+ cells are mainly responsible for HSV-specific cytotoxic activity in some donors, whereas TCR-alpha/beta+ CTL are primarily involved in other subjects. The large majority of HSV-specific CTL bearing either TCR-gamma/delta or TCR-alpha/beta also express CD8 and/or CD56 molecules. Virus-specific CTL, here described, require the expression of HLA class I Ag on the surface of target cells to mediate lytic activity. Nevertheless, the response is apparently HLA-unrestricted in that HSV-1-induced CTL are also able to lyse target cells mismatched for A, B, C, DR, and DQ loci. Our data suggest that both TCR-gamma/delta+ and TCR-alpha/beta+ CTL may play a role in the immune response to HSV in humans.