L-Selectin is a calcium-dependent mammalian lectin that mediates lymphocyte trafficking by recognizing sialylated ligands on high endothelial venules in lymph nodes. Although L-selectin probably mediates neutrophil extravasation into nonlymphoid tissues, no corresponding ligand has been characterized. Staining of cultured endothelial cells with an L-selectin chimera (LS-Rg) showed an internal pool of ligands. Metabolic labeling with sulfur-35-labeled sulfate revealed heparin lyase-sensitive ligands that bound LS-Rg in a calcium-dependent, sialic acid-independent manner. A fraction of commercial heparin bound to LS-Rg and LS-Rg bound to heparin-agarose, both in a calcium-dependent manner. Thus, L-selectin recognizes endothelial heparin-like chains, which could be physiological ligands mediating leucocyte trafficking.