Leukocyte rolling, an early and important step in the inflammatory response, is mediated by the selectin family of adhesion molecules. The selectins bind with low affinity to sialylated and fucosylated glycans such as sialyl Lewisx (sLex), but bind with high affinity to only a few specific glycoproteins on cell surfaces. One such glycoprotein is P- selectin glycoprotein ligand-1 (PSGL-1). The relative contributions of low- and high-affinity ligands to leukocyte rolling in vivo are unknown. We show here that a monoclonal antibody to PSGL-1 (PL1) dramatically reduces rolling of human polymorphonuclear neutrophils (PMN) and promyelocytic HL-60 cells in venules of acutely exteriorized rat mesentery. Control PMN and HL-60 cell rolling flux fractions were 39% +/- 3% and 33% +/- 5%, which were reduced by PL1 to 7% +/- 2% and 6% +/- 2%, respectively. Similar reductions were seen with F(ab) fragments of PL1. PL1-treated PMN rolled at significantly higher mean velocities than untreated PMN owing to intermittent rather that continuous interactions. These findings show that interaction of P- selectin with PSGL-1 is required for rolling of myeloid cells in mesenteric venules at physiologic shear stress in vivo.