Five distinct antigenic sites recognized by cytotoxic T lymphocytes in H-2b mice have been identified on simian virus 40 (SV40) T antigen. Cocultivation of SV40-transformed cells with cytotoxic T lymphocyte (CTL) clones specific for epitopes I–IV results in the selection of variants which show a loss of either epitopes I, II, and III, II and III, or IV. We now report the isolation of a variant SV40-transformed cell line which has lost the expression of all five CTL epitopes on SV40 T antigen. These variant cells were no longer susceptible to lysis by CTL clones specific for all five epitopes. These variant cells would be used to relate alterations in SV40 T antigen structure with functional changes in the transformed cell