It has previously been demonstrated that spleen cells from mice immunized with reovirus type 1 or 3 generate virus-specific cytotoxic T lymphocytes (CTL) after in vitro restimulation. Such cytotoxic T cells lyse H-2 identical targets that are infected with the appropriate reovirus type. Viral recombinants were used to demonstrate that the S1 gene is the predominant viral gene determining the specificity of the CTL. Reoviruses are nonenveloped, non-membrane-maturing viruses; therefore, it was important to determine whether viral products were being recognized by CTL on the surface of target cells. Antiserum blocking was utilized to investigate this issue. Using viral recombinants and antisera to reoviruses types 1 and 3, we were able to demonstrate that the major viral antigen recognized by the CTL on the target cell surface is the σ1 polypeptide encoded by the S1 genome segment. Thus, viral antigens on the target cell membrane seem to be important in the CTL response to a nonenveloped, non-membrane-maturing virus.