Since the preliminary analyses of the glycolipids of small cell carcinomas of the lung showed an increase of GM2 ganglioside, we generated new murine monoclonal antibodies directed to GM2 to identify the molecular species of the glycolipid. The monoclonal antibodies MK2-34 and MK1-16 (both IgM), which specifically detect N-glycolyl GM2 and N-acetyl GM2, respectively, were generated by immunizing mice with liposomes containing monophosphoryl lipid A, trehalose dimycolate, and the antigenic ganglioside. Among the glycolipid preparations extracted from the cancer tissues of 39 patients with lung cancer, a significant amount of N-acetyl GM2 was detected with MK1-16 antibody in 70% of the squamous cell carcinoma cases, 50% of the lung adenocarcinoma cases, 33% of the large cell carcinoma cases, and 100% of the cases of small cell carcinoma of the lung. On the other hand, N-glycolyl GM2 which was defined by the monoclonal MK2-34 was not found in any of the glycolipid fractions prepared from the lung cancer tissues examined in this study. Immunohistochemical studies of the lung cancer tissues with the MK1-16 antibody showed that the N-acetyl GM2 was present not only in small cell carcinoma tissues as one of the antigens related to tumors of neuroectodermal origin, but also in the squamous cell carcinoma and adenocarcinoma of the lung with a comparable frequency. The appearance of the N-acetyl GM2 antigen correlated well with the degree of differentiation of the cancer cells in patients with squamous cell carcinoma and adenocarcinoma of the lung.