P-selectin glycoprotein ligand-1 (PSGL-1) has been proposed as an important tethering ligand for E-selectin and is expressed at a modest level on human leukocytes. Sialyl Lewis x (sLe(x))-like glycans bind to E-selectin and are expressed at a relatively high level on circulating leukocytes. It is unclear whether PSGL-1 has unique biochemical attributes that contribute to its role as an E-selectin ligand. To probe this issue, we conjugated microspheres with either sLe(x) or PSGL-1 purified from myeloid cells (neutrophils and HL-60) and compared their adhesion to endothelial expressed E-selectin under defined shear conditions. We found that both sLe(x) and PSGL-1 microspheres adhere to 4 h of IL-1beta-activated human umbilical vein endothelial cells predominantly through E-selectin. Analysis of the adhesion revealed that the rate of initial tethering of the PSGL-1 microspheres to E-selectin was significantly greater than the rate of initial tethering of the sLe(x) microspheres despite the fact that the sLe(x) microspheres tested had higher ligand densities than the PSGL-1 microspheres. We also found that pretreatment of the PSGL-1 or sLe(x) microspheres with HECA-452 had no significant effect on initial tethering to E-selectin. These results support the hypotheses that 1) PSGL-1 is a high-efficiency tethering ligand for E-selectin, 2) ligand biochemistry can significantly influence initial tethering to E-selectin, and 3) PSGL-1 tethering to E-selectin can occur via non-HECA-452 reactive epitopes.