The use of tumor-infiltrating lymphocytes in the treatment of central nervous system (CNS) neoplasms has met with serious obstacles due to difficulty of culture and poor characterization. Since in other tumors the therapeutic effects of tumor-infiltrating lymphocytes have been shown to rely on T-cell receptor engagement, the authors addressed the question as to whether expression of T-cell receptor variable (V) domains in cultured tumor-infiltrating lymphocytes from CNS is different from that of autologous cultured peripheral blood mononuclear cells. Infiltrating lymphocytes from CNS neoplasms, including primary malignancies, metastatic cancers, and meningiomas, were cultured in the presence of interleukin-2 and anti-CD3 monoclonal antibodies (MoAb's) in order to obtain optimum growth of T cells. Autologous peripheral blood mononuclear cells from the same patients were similarly cultured. After 4 to 5 weeks of culture, 97.3% +/- 2.6% (mean +/- standard deviation) of the resulting cell populations were CD3-positive lymphocytes. The expression of T-cell receptor V domains was then studied by using a panel of 12 MoAb recognizing gene products from T-cell receptor V-alpha 2, V-beta 5, 6, 8, and 12, V-gamma 4 and 9 families, and from two subfamilies of V-delta 2. Remarkably, in over 70% of all paired measurements, percentages of T cells expressing discrete T-cell receptor V-gene products were found to be virtually identical in tumor- and peripheral blood-derived cultured cell populations, with differences never exceeding 1%. In contrast, a different expression of individual V-gene products, concerning both alpha/beta and gamma/delta T-cell receptors, could be detected between cultured tumor-infiltrating lymphocytes and autologous peripheral blood-derived T lymphocytes in seven of 12 patients. In two cases, significant differences between the two populations were also observed in the proliferative responses obtained upon stimulation with staphylococcal enterotoxins that trigger defined V-beta T-cell receptors. Altogether, these data suggest that the T-cell receptor repertoire of cultured tumor-infiltrating lymphocytes from CNS tumors, suitable for use in adoptive immunotherapies, differs from that of autologous cultured peripheral blood mononuclear cells.