Selectin-dependent rolling is the earliest observable event in the recruitment of leukocytes to inflamed tissues. Several glycoproteins decorated with sialic acid, fucose, and/or sulfate have been shown to bind the selectins. The best-characterized selectin ligand is P-selectin glycoprotein-1 (PSGL-1) that supports P-selectin– dependent rolling in vitro and in vivo. In vitro studies have suggested that PSGL-1 may also be a ligand for E- and L-selectins. To study the in vivo function of PSGL-1, without the influence of other leukocyte proteins, the authors observed the interaction of PSGL-1–coated microspheres in mouse venules stimulated to express P- and/or E-selectin. Microspheres coated with functional recombinant PSGL-1 rolled in surgically stimulated and tumor necrosis factor alpha (TNFα)-stimulated mouse venules. P-selectin deficiency or inhibition abolished microsphere rolling in surgically and TNFα-stimulated venules, whereas E-selectin deficiency or inhibition increased microsphere rolling velocity in TNFα-stimulated venules. The results suggest that P-selectin–PSGL-1 interaction alone is sufficient to mediate rolling in vivo and that E-selectin–PSGL-1 interaction supports slow rolling.