Endoglycan is a mucin-like glycoprotein expressed by endothelial cells and some leukocytes and is recognized by L-selectin, a C-type lectin important in leukocyte trafficking and extravasation during inflammation. Here, we show that recombinant L-selectin and human T lymphocytes expressing L-selectin bind to synthetic glycosulfopeptides (GSPs). These synthetic glycosulfopeptides contain 37 amino acid residues modeled after the N-terminus of human endoglycan and contain one or two tyrosine sulfates (TyrSO(3)) along with a nearby core-2-based Thr-linked O-glycan with sialyl Lewis x (C2-SLe(x)). TyrSO(3) at position Y118 was more critical for binding than at Y97. C2-SLe(x) at T124 was required for L-selectin recognition. Interestingly, under similar conditions, neither L-selectin nor T lymphocytes showed appreciable binding to the sulfated carbohydrate epitope 6-sulfo-SLe(x). P-selectin also bound to endoglycan-based GSPs but with lower affinity than toward GSPs modeled after PSGL-1, the physiological ligand for P- and L-selectin that is expressed on leukocytes. These results demonstrate that TyrSO(3) residues in association with a C2-SLe(x) moiety within endoglycan and PSGL-1 are preferentially recognized by L-selectin.