Neutrophil tethering and rolling in shear flow are mediated by selectins and have been thought to be two indistinguishable manifestations of a single molecular interaction between selectin and ligand. However, we report that under physiologic flow conditions, tethering to E-selectin requires a ligand distinct from the one that supports neutrophil rolling. Tethering under shear to E-selectin requires a carbohydrate ligand that is closely associated with the lectin domain of L-selectin on the neutrophil surface, as enzymatic removal of L-selectin, chemotactic factor-induced shedding of L-selectin, and L-selectin MAbs effectively block tethering. In contrast, this ligand is dispensable for the ability to roll on E-selectin, since rolling adhesions formed after static incubations were not affected by the presence or absence of L-selectin. Thus, E-selectin interactions with ligands on neutrophils persist after L-selectin shedding. These findings add an additional step for regulation of leukocyte localization in inflammatory sites.