Although the extensive family of non-H-2 histocompatibility (H) antigens provides a formidable barrier to transplantation, the origin of their encoding genes are unknown. Recent studies have demonstrated both the linkage between H genes and retroviral sequences and the ability of integrated Moloney-murine leukemia virus to encode what is operationally defined as a non-H-2 H antigen. The experiments described in this communication reveal that skin grafts from an SV40 T-antigen transgenic C57BL/6 mouse strain are rejected by coisogenic C57BL/6 recipients with a median survival time of 49 days, which is comparable to those of many previously defined non-H-2 H antigens. The specificity of this response for SV40 T-antigen was demonstrated by the identification of SV40 T-antigen-specific cytolytic T lymphocytes and antibodies in multiply-grafted recipients. Although these cytolytic T lymphocytes could detect SV40 T-antigen on syngeneic SV40-transformed fibroblasts, they neither could be stimulated by splenic lymphocytes from T-antigen transgenics nor could they lyse lymphoblast targets from T-antigen transgenics. These observations suggest a limited tissue distribution of SV40 T-antigen in these transgenics. These results confirm the role of viral genes in the determination of non-H-2 histocompatibility antigens by the strict criteria that such antigens stimulate (1) tissue graft rejection and (2) generation of cytolytic T lymphocytes. Furthermore, they suggest that the SV40 enhancer and promoter region can target expression of SV-40 T-antigen to skin cells of transgenic animals.