Previous studies suggested that sialosyl-Le(x) (SLex) is a ligand expressed in human neutrophils and myelogenous leukemia HL60 cells which binds to E-selectin and possibly P-selectin. However, clear data on structures of carbohydrate epitopes in these cells were lacking. A systematic study was therefore initiated, employing a large quantity of HL60 cells (> or = 1200 mL packed) and human leukocytes (approximately 100 mL packed). Gangliosides were extracted, followed by extensive fractionation and examination of the E- and P-selectin binding ability of each fraction. The following results were of particular interest: (i) Only monosialogangliosides having a polylactosamine core with > 10 monosaccharide units (or > 4 N-acetyllactosamine units) showed E-selectin binding under static conditions with thin-layer chromatography overlay technique employing 32P-labeled E-selectin-expressing CHO cells. (ii) Sulfate groups were not detectable in the binding fractions, and di- and trisialoganglioside fractions did not show E-selectin binding under these conditions. (iii) None of the fractions showed P-selectin binding under a similar assay system using 32P-labeled P-selectin-expressing CHO cells. (iv) Major gangliosides of HL60 cells were structures I-XI (shown in Table 1 of text), none of which showed E-selectin binding under the above conditions. (v) SLex gangliosides having tetraosyl to octaosyl ceramide core, which are the major gangliosides of epithelial tumors (shown in Table 2), were completely absent from HL60 cells and neutrophils. Isolation and chemical characterization of ganglioside structures I-XI are described in this paper.