Neutral glycosphingolipids were isolated from the malignant cells of several patients with different types of acute leukemia. Analyses were performed by high performance liquid chromatography combined with enzymatic hydrolysis of glycosphingolipids using glycosidases (Escherichia freundii endo-beta-galactosidase, jack bean beta-galactosidase, and beef kidney beta-hexosaminidase). We found that acute leukemia cells contain very little or none of the more complex neutral glycosphingolipids that are found in normal leukocytes or chronic leukemia cells. Lymphoblasts, in particular, are rich in neutral glycosphingolipids with only 1 or 2 carbohydrate units. The most significant finding of our study was that, in contrast to normal leukocytes and chronic leukemia cells which have a single predominant tetraosylceramide species, acute leukemia cells (9 out of 10 patients analyzed) were found to have significant amounts of both globo (GalNAc beta 1 leads to 3Gal alpha 1 leads to 4Gal beta 1 leads to 4Glc beta 1 leads to 1ceramide) and neolactotetraosylceramide (Gal beta 1 leads to 4GlcNAc beta 1 leads to 3Gal beta 1 leads to 4Glc beta 1 leads to 1ceramide). These results indicate that the composition of neutral glycosphingolipids in acute leukemia cells differs significantly from that found in normal or chronic leukemia cells.