We previously demonstrated that intratumoral administration of liposomes containing the murine interferon beta (IFN-beta) gene [lip(pSV2muIFN-beta)] resulted in stronger growth-inhibitory effect on GL261 (H-2b) mouse glioma inoculated in brains of syngeneic C57BL/6 mice than conventional exogenous IFN-beta administration, and histologic evaluation revealed the massive infiltration of T lymphocytes (CD8 > CD4) within the residual tumor. The present study was aimed at determining whether such tumor-infiltrating lymphocytes (TIL) have any tumor-specific cytotoxic effects. Intratumoral administration of lip(pSV2muIFN-beta) resulted in prolonged survival time and a 50% tumor-free incidence in the mice treated. The surviving animals were subsequently re-challenged with either subcutaneous or intracranial injection of GL261 cells, and no tumors were found to develop over a 50-day period. In vivo depletion of CD8, but not CD4 cells decreased the efficacy of lip(pSV2muIFN-beta). Specific cytotoxic T lymphocytes (CTL) against GL261 cells were generated from both TIL and spleen cells of the mice treated. The results of flow cytometric analysis and antibody blocking test revealed that the bulk CTL lines thus prepared were T cell receptor (TCR) alphabeta, CD8 T lymphocytes with H-2b restriction. These findings suggest that, in addition to direct growth-inhibitory effects by the IFN-beta gene on the tumor cells, activation of systemic cellular immunity may participate in antitumor effects in vivo, despite the fact that central nervous system is generally regarded as an immunologically privileged site.