Cytotoxic T lymphocytes (CTL) were generated in C57BL/6 mice with herpes simplex virus type 1 (HSV-1) (strains KOS, 17, HFEM, and mP) and HSV-2 (strains 186, G, and GP6). Effector lymphocytes were tested for cytotoxicity against syngeneic HSV-1- and HSV-2-infected cells in a 5-h 51Cr release assay. HSV-1 strain HFEM was found to induce CTL efficiently only when 100-fold more virus was used as compared with HSV-1 strains KOS, 17, and mP. All HSV-1 and HSV-2 strains induced cross-reactive populations of CTL. CTL generated by HSV-1 KOS and HSV-2 186 also demonstrated cross-reactivity in an ear-swelling model for delayed-type hypersensitivity. Lymphocytes generated by all HSV-2 strains were highly efficient at lysing HSV-1-infected target cells. However, HSV-2-infected target cells were found to be less susceptible to lysis by either HSV-1 or HSV-2 CTL than were HSV-1-infected target cells. The lowered susceptibility of HSV-2-infected cells was not due to an inefficient infection of BL/6 WT-3 cells as measured by standard growth assays and infectious center assays. Varying the multiplicity of infection or the time of infection did not increase the susceptibility of HSV-2-infected target cells to lysis by CTL. Increasing the effector-to-target-cell ratio resulted in an increased lysis of both HSV-1- and HSV-2-infected target cells by CTL, but the level of HSV-2-infected target cell lysis still did not approach the level of HSV-1-infected target cell lysis. HSV-2-infected cells were as efficient as HSV-1-infected cells in the cold cell competition assay employed in reducing the lysis of 51Cr-labeled, HSV-1-infected target cells. In addition, HSV-2-infected cells were susceptible to lysis by HSV-immune serum and complement.