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 if 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 HL60) 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 hr. IL-1 activated human umbilical vein endothelial cells (HUVEC) 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 pre-treatment 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 (i) PSGL-1 is a high efficiency tethering ligand for E-selectin, (ii) ligand biochemistry can significantly influence initial tethering to E-selectin and (iii) PSGL-1 tethering to E-selectin can occur via non-HECA-452 reactive epitopes.