The nature of the host's immune response to isografts of hamster embryo fibroblasts (HEF) transformed by herpes simplex virus type 1 (HSV-1) was investigated by the microcytotoxicity assay. It was found that spleen cells from tumor-bearing hamsters killed homologous tumor cells but not HEF transformed by cytomegalovirus or PARA-(defective SV40)-adenovirus 7 (PARA-7). Cytotoxicity was lost as the tumor increased in size. Spleen cells from animals bearing isografts of HSV type 2 (HSV-2) transformed cells also killed HSV-1 target cells whereas spleen cells from PARA-7 tumor bearers did not. Further studies showed that animals immunized with HSV-1 or HSV-1-infected rabbit kidney cells produced spleen cells specifically cytotoxic for HSV-transformed cells. On the other hand, sera from virus-immunized hosts or tumor bearers had no effect on the cells in the presence of guinea pig complement. However, in blocking experiments such sera could significantly reduce spleen cell cytotoxicity. These experiments established that cells transformed by HSV could elicit a cellular immune response in the syngeneic host, and provided evidence that the immunity was directed against virus-specific antigens on the cell surface.