Previous studies in mice revealed that resolving intraocular tumors (UV5C25 fibrosarcoma) were infiltrated with mononuclear cells and invoked potent systemic delayed-type hypersensitivity responses without nonspecific tissue destruction. The present study characterized the tumor-infiltrating lymphocyte (TIL) population and established its role as the mediator of specific intraocular tumor rejection. This was accomplished by (a) isolating TIL from resolving intraocular tumors; (b) identifying characteristic surface markers on TIL; and (c) demonstrating in vitro and in vivo antitumor functions. Fluorescence-activated cell sorter analysis of TIL showed 33.4% Thyl+, 19.8% CD8+, 11.1% CD4+, 17.2% MAC-1+, 10.4% F4/80+, and 7.7% B220+. Functional studies indicated that TIL were directly cytolytic for UV5C25 tumor cells. Additionally a tumor-necrosis-factor(TNF)-sensitive cell line (WEHI 164.1) was lysed on cocultivation with TIL, whereas UV5C25 tumor cells were insensitive to lysis by TNF. Precursor CTL analysis demonstrated a high frequency (1/251) of tumor-specific precursors and a low frequency of alloresponsive cells in the TIL population. In vivo analysis by a Winn-type assay demonstrated that only TIL could effect tumor resolution in immunosuppressed hosts. These results demonstrate that although CD4+ T cells and macrophages were present and TNF activity was detected in the TIL population, there was no evidence for nonspecific tissue destruction within the eye. Therefore, this pattern of intraocular tumor rejection is mediated by a lymphocyte population expressing cell-surface phenotypes and functional characteristics of conventional cytotoxic T lymphocytes. Moreover, the results suggest that a regulatory mechanism within the eye allows for the emergence of one dominant antitumor effector (CTL) while controlling a more destructive mechanism (delayed-type hypersensitivity).