Operationally specific monoclonal antibodies (MAbs) reactive with tumor but not normal adult tissues offer great potential for diagnosis and therapy of CNS neoplasms. Two targets for specific MAb localization were chosen for this study: (1) glioma-associated gangliosides GM2 [II3NeuAc-GgOse3Cer], GD2 [II3(NeuAc)2-GgOse3Cer], GD3[II3(NeuAc)2-LacCer], 3'-isoLM1 [IV3NeuAc-LcOse4Cer], and 3',6'-isoLD1 [IV3NeuAc,III6NeuAc-LcOse4Cer] and (2) epidermal growth factor receptor (EGFR) variant molecules. Epitopic specificity of isolated ganglioside hybridomas was determined with FAB-MS defined ganglioside standards. All MAb are IgM. Assay of 14 cytologic specimens and 31 frozen sections of primary CNS neoplasms revealed staining with anti-GD3 (14/14, 31/31), anti-GM2 (9/14, 26/31), and anti-GD2 (6/14, 24/30), respectively. 3'-isoLM1 and 3',6' isoLD1, which exhibit a restricted oncofetal expression pattern and are not detectable in adult human brain, are present in 15/31 primary CNS neoplasms and in 1/8 human glioma xenografts, as detected by MAbs SL-50 and DMAb-14, respectively. EGFR proteins, the second target, have unique amino acid spans resulting from gene deletion in the amplified EGFR gene present in subsets of malignant human gliomas. Antibodies against EGFR deletion-mutant Type III show highly restricted activity with a subset of glioma biopsies (6/35) expressing the mutant EGFR. These reagents should be useful for in vitro and in vivo diagnosis and, potentially, for treatment of malignant brain tumors.