In search of endogenous oligosaccharide ligands for the endothelial adhesion molecule E-selectin in mouse, glycolipids from tissues and the neutrophilic cell line 32D c13 were tested for E-selectin binding. Kidneys of BALB/c and NMRI mice (but not CBA) and the 32D c13 cells were found to contain minor glycolipid populations that support strongly the binding of murine E-selectin. By chromatogram binding experiments and in situ liquid secondary ion mass spectrometry (LSIMS) with neoglycolipids derived from their endoglycoceramidase-released oligosaccharides, in conjunction with compositional and linkage analyses, one of the glycolipid ligands in kidney was identified as the Le(x)-active extended globo-glycolipid: [formula: see text] Neoglycolipids enriched for the ligand structures were obtained from oligosaccharides released by endo-beta-galactosidase from the 32D c13 cells. By TLC-LSIMS and antibody binding, the main E-selectin binding determinant on these was identified as sialyl-Le(a).