Brain tumors carry a poor prognosis, and newer approaches to their therapy are urgently needed. Natural killer T (NKT) cells are distinct innate lymphocytes with antitumor potentials. Defects in NKT cell function have been observed in patients with other forms of cancer. Here we show that both the frequency and interferon-γ-producing function of NKT cells are well preserved in adult patients with glioma (n = 9) and comparable to findings in healthy controls (n = 9). These cells can be readily expanded in culture using autologous mature dendritic cells loaded with the NKT ligand, α-galactosyl ceramide. The expanded NKT cells from glioma patients are functional and, importantly, kill glioma cells in a ligand- and CD1d-dependent manner. Expression of CD1d is detected both on primary glioma cells as well as endothelial cells in infiltrating new blood vessels by immunohistochemistry of glioma tissue sections. These data suggest that targeting NKT cells may provide a novel strategy for immunotherapy of glioma. © 2004 Wiley-Liss, Inc.