Human papovavirus 1C virus (JCV) and Simian virus 40 (SV40) tumor or T antigens demonstrate considerable sequence homology which is reflected by antibody cross-reactivity. This similarity raised the possibility that JCV and SV40 T antigen also might contain common cytotoxic T lymphocyte (CTL) recognition epitopes. In this study we identified and mapped such sites on the JCV T antigen. C57BI/6 cell lines transformed by JCV/SV40 T antigen chimeras were generated and tested for susceptibility to lysis by five H-2b restricted SV40-specific CTL clones: Y-1, Y-2, Y-3, Y-4, and Y-5. These CTL clones recognize specific epitopes within amino acids 205-219 (site I), 220-233 (sites 11 and III), 369-511 (site IV), and 489-503 (site V) on SV40 T Ag, respectively. The results show that sites I, 11, III, and IV (recognized by CTL clones Y-1, Y-2, Y-3, and Y-4, respectively) represent common epitopes on SV40 and JCV T antigens. CTL clone Y-5 failed to recognize JCV T antigen indicating that CTL can discriminate between the two antigenically related T antigens. © 1991.