Monoclonal antibodies (mAbs) recognizing the disialoganglioside II3(NeuAc)2GgOse3Cer (GD2) were produced by immunizing mice with the GD2-expressing neuroblastoma cell line LAN-1 and a prefusion boost with purified GD2 coupled to Salmonella minnesota. Two IgM mAbs were isolated which demonstrated high levels of reactivity (binding ratios in excess of 100) with GD2 by solid-phase radioimmunoassay and positivity in high-performance thin-layer chromatography (HPTLC) immunostain; only one (DMAb-20) was subsequently shown by analysis with a panel of defined ganglioside species to be specific for the minimum epitope of GD2 GalNAc beta 1-4(NeuAc alpha 2-8-NeuAc alpha 2-3)Gal-, DMAb-20 was used to evaluate the expression of GD2 by malignant glioma and medulloblastoma cell lines using cell surface radioimmunoassay. indirect membrane immunofluorescence. HPTLC immunostain, and densitometric analysis of extracted gangliosides from selected cell lines. Sixteen of 20 (80%) malignant glioma and 5 of 5 medulloblastoma cell lines reacted with DMAb-20; in agreement with previous studies, 5 of 5 neuroblastoma and 2 of 3 melanoma cell lines also reacted with DMAb-20, GD2 was proportionally increased in the glioma and medulloblastoma cell lines relative to levels in normal brain, as determined by densitometric analysis. In a phenotypic survey of malignant glioma biopsies, tumor cells in 24 of 30 (80%) cases stained positively with DMAb-20. Reactive astrocytes, both within the adjacent to tumors, were frequently intensely stained. Among the morphological variants of glioblastoma examined, the most intense staining with DMAb-20 was observed in neoplastic gemistocytes, with the weakest or absent staining in small cell glioblastomas. As GD2 is a commonly expressed surface antigen of gliomas and medulloblastomas, expression of which is retained in tissue culture. DMAb-20 will be useful in determining the functional role of GD2 in cell-cell interaction, adhesion, and invasion, and in defining altered growth control mechanisms of central nervous system neoplasms in in vitro models.