A series of synthetic oligosaccharides based on sialyl Lewis x (sLex; Neu5Ac alpha 2-3Gal beta 1-4[Fuc alpha 1-3]GlcNAc) and sialyl Lewis a (sLea; Neu5Ac alpha 2-3Gal beta 1-3[Fuc alpha 1-4]GlcNAc) was used to study the binding interactions of selectins. E-selectin-immunoglobulin fusion protein (E-selectin-Ig) bound to immobilized bovine serum albumin (BSA)-neoglycoproteins containing sLex or sLea in a Ca(2+)-dependent manner. Solution-phase sLex tetrasaccharide blocked this interaction by 50% at a concentration of 750 +/- 20 microM (IC50). sLea was more effective (IC50 = 220 +/- 20 microM), while nonsialylated, nonfucosylated derivatives showed little or no activity at concentrations up to 1 mM. Attachment of an 8-methoxycarbonyloctyl aglycone in a beta linkage to the anomeric carbon of the GlcNAc of sLex or sLea increased their blocking activity nearly twofold. Finally, replacement of the 2-N-acetyl substituent of the GlcNAc by an azido or amino group resulted in substantial increases in activity, with the most potent inhibitor being amino substituted sLea, which was 36-fold more active (IC50 = 21 +/- 3 microM) than the reducing tetrasaccharide sLex. In contrast to results obtained with E-selectin-Ig, P-selectin-Ig binding to immobilized BSA-sLea was blocked modestly by most oligosaccharides at 1 mM, with no substantial differences among them. IC50 values of soluble oligosaccharides determined in competitive binding studies accurately predicted blocking of leukocyte adhesion to recombinant E-selectin-Ig and to cytokine-activated endothelium.