Selectins constitute a family of proteins that mediate leukocyte tethering and rolling along the vascular endothelium by recognizing various carbohydrate ligands in response to inflammation. To test the hypothesis that multivalent binding of selectins to their ligands is the molecular basis for achieving sufficient binding forces, we have performed this flow chamber study. Selectin-containing Chinese hamster ovarial cells (CHO-E) bind and roll along a support-fixed phospholipid membrane containing a defined concentration of a synthetic Sialyl Lewisx (sLex) glycolipid ligand. Ligands are either homogeneously distributed, or arranged in defined lateral clusters, as illustrated here for the first time. The lateral glycolipid clusters which appear as recognition motifs are essential for mediating cell rolling. Furthermore, the transition from firm cell adhesion to cell rolling depends on the site density of ligands. Rolling velocity shows little dependence on shear forces within a broad range. As we found out that cells do not roll along the model membranes with homogeneous ligand distribution, our results therefore support the hypothesis of multivalent binding events. Since these investigations suggest that lipid-anchored sLex, functionally embedded in a lipid matrix, can mediate cell rolling, this study demonstrates the relationship between dynamic glycolipid binding to selectins with the hypothesis of multivalency of binding for the first time.