It has been shown that adoptive immunotherapy can be curative for established malignant tumors. The key to this treatment lies in obtaining sufficient numbers of lymphocytes which are sensitized to recognize tumor antigens and carry out immunological reactions to destroy tumor cells. Reported here are the results of experiments to: 1) sensitize lymphocytes to the antigens of rat glioma cells and expand them ex vivo for use in adoptive immunotherapy, 2) characterize the cells of the expanded population, and 3) evaluate antitumor activity in a cohort of rats with well-established intracranial gliomas. Viable RT-2 glioma cells were injected into the hind foot pads of syngeneic Fischer 344 rats. After 10 days, the tumor draining lymph nodes (DLN) were harvested from the injected limbs and mechanically dissociated. The cells of the DLN were then suspended in culture medium supplemented with low dose interleukin-2 (IL-2) and incubated for 18 hours with Bryostatin-1 and ionomycin (Bryo/Io) to stimulate expansion. The cells were next washed to remove the Bryo/Io and resuspended in culture medium and IL-2. Population expansions of 40- to 100-fold were seen after 8 days. Flow cytometric analysis showed these cells to be a nearly pure population of T lymphocytes of the CD3+CD8+ phenotype. Intravenous injection of the ex vivo expanded DLN cells did not significantly improve survival of rats with a seven-day intracerebral RT-2 glioma, although, compared to untreated controls, the tumors of the treated animals were smaller, showed no necrosis, and appeared to be less infiltrative. Furthermore, the treated animals had a pronounced lymphocytic infiltration of their tumors with greater associated degrees of hemorrhagic change and peritumoral edema. When the ex vivo expanded DLN cells were intravenously injected into three-day intracerebral RT-2 glioma models, tumors were almost always eliminated and the animals survived their tumor challenge. We conclude that successful expansion of glioma-sensitized DLN lymphocytes is possible and that adoptive immunotherapy using these cells is capable of effectively limiting the progression of large gliomas, while totally eradicating small ones.