Recent reports have documented the presence of SV40 large T antigen (T ag) sequences in a number of human tumors and raised the question of whether cellular immunity to T ag is elicited in such individuals. We used HLA-A2.1 transgenic C57BL/6 mice to identify an epitope from T ag recognized by CD8+ CTLs when presented by this human MHC class I molecule. Immunization of HLA-A2.1 transgenic mice with syngeneic T ag-transformed cells resulted in the induction of HLA-A2.1-restricted, T ag-specific CTLs. The target epitope, residues 281-289 (KCDDVLLLL) of T ag, was identified using both cell lines expressing T ag variants and synthetic T ag peptides. Peptide 281-289 bound stably to HLA-A2.1 molecules, effectively sensitized target cells for CTL lysis, and was efficiently processed from endogenous T ag in cells of both mouse and human origin. CTLs were not cross-reactive on the human BK or JC virus T ags. Thus, SV40 T ag 281-289 represents a potential specific CTL recognition epitope for humans.