Tumor-infiltrating lymphocytes (TIL) were grown from 23 urothelial carcinomas. Phenotyping analysis showed that the TIL cultures were mainly CD3 1 . Although CD4 1 and CD8 1 T-cell sub-sets were grown in culture, CD4 1 T-cell sub-sets predominated over CD8 1 T cells. Immunohistochemical studies performed on 5 tumor specimens confirmed this observation, and indicated that CD4 1 T cells surrounded the tumor islets, whereas CD8 1 T lymphocytes were localized among the tumor cells. Five short-term carcinoma cell lines established from these urothelial tumors were used as target cells in cytolysis assays in order to investigate the functional anti-tumor activity of autologous TIL. TIL from 4/5 tumors were lytic and 3 TIL lines displayed MHC-class-I-dependent cytotoxicity directed against autologous tumor cells. CD4 1 T-cell-depletion experiments performed on TIL line 07 confirmed that CD8 1 MHC-class-I-dependent CTL were the predominant effectors. Finally, experiments performed on 6 allogeneic urothelial-cancer cell lines matched for HLAclass-I molecules showed that TIL07 exhibited selective lytic activity toward tumor 07. These data indicate that CD8 1 MHC-class-I-dependent CTL present in urothelial carcinomas are functional and may participate in the anti-tumor immune response.