The initial adhesive interactions between circulating leukocytes and endothelia are mediated, in part, by P-selectin. We now report the expression cloning of a functional iigand for P-selectin from an HL-60 cDNA library. The predicted amino acid sequence reveals a novel mucin-like transmembrane protein. Significant binding of transfected COS cells to P-selectin requires coexpression of both the protein iigand and a fucosyltransferase. This binding is calcium dependent and can be inhibited by a neutralfzing monoclonal antibody to P-selectin. Cotransfected COS ceils express the ligand as a homodimer of 220 kd. A soluble ligand construct, when coexpressed with fucosyitransferase in COS cells, also mediates P-se&tin bindlng and is immunocrossreactive with the major HL-60 glycoprotein that specifically binds P-selectin.