We have recently reported that the disialoganglioside GD3 is found in cellular lipid extracts of T-cell acute lymphoblastic malignancies (T-ALL) but is not detectable by resorcinol staining in extracts of non-T acute lymphoblastic leukemia blasts (non-T-ALL). We have now extended this study to assess the detectability of GD3 in T-ALL vs non-T-ALL utilizing an anti-GD3 antibody, R24. Gangliosides isolated from T-ALL and non-T-ALL blasts by two different methods were separated by thin-layer chromatography and stained with anti-GD3 and a control antibody specific for GM3 and sialosylparagloboside (SPG). Anti-GD3 reactivity was observed in extracts from T-ALL cells in all cases, whereas GD3 was not detected in any of the non-T-ALL samples. The anti-GM3/SPG antibody stained GM3 in all of the leukemic samples analyzed as well as SPG in the non-T-ALL samples. Indirect immunofluorescence was used to assess the expression of GD3 at the surface of leukemic blasts. Fluorescence-activated cell sorting analysis with R24 showed that whereas T-ALL blasts were highly reactive with this antibody, non-T-ALL blasts were totally unreactive. In an analysis of a larger number of leukemia patients by fluorescence microscopy, 20 out of 28 samples with the T-ALL phenotype were positive for R24, whereas zero out of 11 non-T-ALL samples were reactive. These results confirm our earlier finding of the specificity of GD3 to the T-ALL subclass of childhood leukemias and furthermore suggest the potential value of anti-GD3 as an immunological tool for the diagnosis and therapy of T-cell ALL.