Tumor-infiltrating lymphocytes (TIL) from six gynecologic malignant tumors (two uterine cervical cancers, two ovarian serous cystadenocarcinomas, and two uterine corpus cancers), cultured in the presence of recombinant interleukin 2, were assayed for their cytotoxic activities against various fresh tumor cells including autologous tumors. A clear correlation between phenotype and cytotoxic activity of TIL was observed. Four of six TIL preparations exhibited strong cytotoxic activity against autologous fresh tumor target cells, and were all CD8+. In contrast, cytotoxic activity was not detected in any of the CD4+ TIL preparations. The cytotoxic activities of the CD8+ TIL preparations were highly specific; only autologous fresh tumor cells were lysed. This result is consistent with the notion that TIL are of a different cell lineage from lymphokine-activated killer cells which are antigen-nonspecific and CD8-. Instead, TIL appear to be of cytotoxic T cell lineage that is highly antigen-specific and CD8+. To explore the potential for clinical use, we have attempted to augment the cytotoxic activities of these CD8+ TIL by treatment of the target tumor cells with gamma interferon (IFN) in vitro, hoping that elevated expression of MHC class I gene products on the cell surface would enhance their recognition. It was observed that brief treatment of freshly prepared tumor cells in vitro with gamma-IFN resulted in augmentation of the expression of MHC class I gene products, and the treated tumor cells were more susceptible to lysis by TIL than untreated cells.