P-selectin glycoprotein ligand 1 (PSGL-1) is a mucinlike glycoprotein expressed on the surface of myeloid cells and serves as the high affinity counterreceptor for P-selectin. The PSGL-1-P-selectin interaction is calcium dependent and requires presentation of sialyl-Lewis”(sLe”)-type structures on the O-linked glycans of PSGL-1. We report here the identification of a noncarbohydrate component of the binding determinant that is critical for high affinity binding to P-selectin. Located within the first 19 amino acids, this anionic polypeptide segment contains at least one sulfated tyrosine residue. We propose that this sulfotyrosinecontaining segment of PSGL-1, in conjunction with sLe” presented on O-linked glycans, constitutes the high affinity P-selectin-binding site.