The glycosphingolipids GD3, GM3, and alpha 2, 3-sialosylparagloboside (SPG) are major gangliosides of lymphoid leukemia cells. The reactivity of two monoclonal anti-ganglioside antibodies, an anti-GD3 (R24) and an antibody cross-reactive to GM3 and SPG (M2590), to blasts of patients with T-cell acute lymphoblastic leukemia (ALL) and B-cell precursor ALL (pre-B-ALL), were compared using indirect immunofluorescence and flow cytometry. Results from 23 patients with T-ALL and eight with pre-B-ALL yielded four subclasses of T-ALL and two subclasses of pre-B-ALL. Blasts from most of the patients with T-ALL were R24+M2590- whereas most of the patients with pre-B-ALL were R24-M2590-. Seven of 23 patients with T-ALL had ganglioside immunophenotypes similar to that of pre-B-ALL, i.e. R24-M2590- or R24-M2590+. These subclasses could not be further characterized by additional cell surface immunophenotypic markers or by gene (immunoglobulin and T-cell receptor) rearrangement analysis. The ratio R24/M2590 was less than 1.0 in all patients with pre-B-ALL, and was greater than 1.0 in all patients with T-ALL who were R24 positive, but was not useful in characterizing the double negative T-ALL subclass. To assess whether cryptogenicity of gangliosides due to cell surface protein could account for the low binding of either R24 or M2590, blasts were treated with trypsin before antibody analysis. Whereas the binding of R24 was unchanged after trypsin treatment, binding of M2590 was increased in a number of samples, particularly in those samples which were originally M2590-positive. The results show that comparative staining of T-ALL and pre-B-ALL cells with both anti-GD3 and anti-GM3/SPG antibodies results in a further subclassification of ALL and provides a quantitative assessment of the expression of tumor-associated gangliosides on the blasts of this disease.