The present study describes a method for in vitro expansion and characterization of antitumor-reactive lymphoid cells isolated from human maligant astrocytomas. Glioma-infiltrating lymphocytes were separated from 24 glioma specimens and cultured in medium containing interleukin 2 (50 to 2000 units/ml). Within 20 to 42 days after the initiation of culture, 20 of 24 cultures of glioma-derived lymphocytes expanded with a substantial increase in cell numbers, of at least 5 .times. 108 cells up to 5 .times. 109, whith a simultaneous elimination of contaminating autologous gliomas cells. The expanding glioma-derived lymphocytes consisted of 90 .+-. 8% (SD) CD3+ T-cells including both CD4+ and CD8+ subpopulations. CD16 was expressed on 4 .+-. 5% of the cells and three cultures studied exhibited 14% .+-. 1 of Leu-19-positive cells. After 4 to 8 weeks of proliferation, interleukin 2 receptor expression decreased from 36 .+-. 28% to less than 10% and the lymphocytes ceased to grown in all cultures. Glioma-derived effector lymphocytes could lyse almost all the autologous tumor targets as well as allogeneic glioma cells. The cytotoxic activity of long-term cultured peripheral blood lymphocytes obtained from the same patients appeared to be similar to that of glioma-derived lymphocytes in killing autologous tumor cells. In summary glioma-derived lymphocytes expanded in bulk culture with high concentrations of interleukin 2 (2000 units/ml) consisted predominantly of T-lymphoblasts with the ability to kill autologous glioma cells. The tumor-infiltrating lymphocytes could be expanded to sufficient numbers for possible use in the adoptive immunotherapy of malignant gliomas.