The simian virus 40 (SV40) tumor or T antigen synthesized in transformed or infected cells is highly immunogenic, inducing both antibody and cytotoxic T lymphocyte (CTL) responses. In the C57BL/6 (H-2b) strain of mice the CTL response is directed to discrete sites on T antigen. To date, five CTL recognition sites have been identified using CTL clones, deletion mutants and overlapping synthetic peptides. The CTL sites I, II and III are clustered in the amino-terminal one-third of T antigen, whereas sites IV and V are located in the carboxyl one-third. Using synthetic peptides, the site I has been tentatively assigned to residues 205 to 215 of T antigen and sites II and III map to residues 220 to 233. Site V maps to amino acids 489 to 503. The location of site IV remains undefined but probably falls between amino acids 368 and 511. The CTL sites I, II, III and V are H-2Db-restricted, whereas site IV is H-2Kb-restricted. CTL sites II and III can be distinguished using H-2Db class I mutants which present the same peptide differentially to CTL clones specific for sites II and III. The multiplicity of CTL sites on SV40 T antigen contributes to the overall immunosurveillance in the host against SV40 carcinogenesis. In the event of a loss of a particular site due to mutation or deletion, the remaining CTL sites continue to provide an effective target for CTL-mediated surveillance. Similar events may also contribute toward controlling papovavirus infections in humans.