Hybridoma cells were derived from a fusion between mouse P3x63/Ag8 myeloma cells and spleen cells from a mouse immunized with whole cells of a human malignant glioma line. Of 345 hybrids obtained, 36 secreted antibodies that reacted with the glioma cell line used for immunization as assayed by an indirect antibody-binding radioimmunoassay. After a first screening for the absence of reactivity on two nongliogenous cell lines, 3 hybrids were selected and cloned by limiting dilution. The specificity of these monoclonal antibodies was then investigated on a panel of 18 cell lines derived from human malignant gliomas, 18 cell lines from nongliogenous neoplasms, as well as normal peripheral blood lymphocytes, normal skin fibroblasts, and normal spermatozoa. The monoclonal antibodies from two positive hybrids, BF7 and GE2, reacted exclusively with glioma cells and appeared to be directed against common malignant glioma antigen(s). BF7 antibodies bound to 13 and GE2 to 17 of 18 glioma cell lines. The third monoclonal antibody, CG12, showed a broad reactivity since it bound to 10 of 18 glioma lines, five of five melanoma lines, and one of one neuroblastoma line. Absorption with normal adult and fetal brain homogenate did not modify the binding capacity of BF7 and GE2 for glioma cells, while the binding of CG12 antibodies was abolished. Reciprocal binding inhibition tests using [3H]leucine-labeled antibodies showed that BF7, GE2, and CG12 antibodies were directed against different antigenic determinants.