The purpose of the present study was to define the usefulness of limiting dilution analysis (LDA) to enumerate glioma-reactive cytolytic T lymphocytes (CTL) as a constituent of tumor infiltrating lymphocytes (TIL) isolated from rat gliomas. Optimum LDA microculture conditions were defined by co-cultivating graded numbers of responder TIL together with 10(5) irradiated syngeneic rat splenocytes, 10(3) irradiated glioma cells, and 10 U/well of recombinant interleukin-2 incubated for 8 days. Antigenic specificity of the anti-tumor response was demonstrated by high levels of [3H]thymidine incorporation by TIL derived from F98 gliomas following stimulation with irradiated F98 glioma cells compared to low levels following stimulation with the antigenically distinct D74 glioma cells. Limiting dilution analysis showed that cytolytic T lymphocyte-precursors were present in TIL of F98 gliomas of immunized rats at an approximate frequency of 300 CTL/10(6) TIL, indicating that less than 1% of the TIL were tumor-reactive CTL. As determined by cell depletion experiments using various MoAbs and complement, the majority of the cytolytic activity detected against glioma targets was mediated by OX-8+ TIL. Split culture experiments revealed that high levels of glioma-reactive CTL activity and low levels of NK activity, which are simultaneously detected among TIL, were mediated by separate cell populations. Our data demonstrate that LDA microcultures can be used as a powerful tool to differentiate tumor-reactive CTL from other effector cell populations.