Lectin-carbohydrate recognition between the selectins and their ligands are among the earliest events in leukocyte recirculation, leukocyte recruitment into inflamed areas, and abnormal egress of leukocytes in diseases. Previously, we have described a dimeric sialoglycoprotein from myeloid cells with subunits of molecular mass = 120 kDa, which is selectively recognized by P-selectin (Moore, K.L., Stults, N.L., Diaz, S., Smith, D.F., Cummings, R.D., Varki, A., and McEver, R.P. (1992) J. Cell Biol. 188, 445-456). Here, we demonstrate that this P-selectin ligand carries alpha 2-3-linked sialic acids and the sialyl-Lewisx (SLex) tetrasaccharide motif. This glycoprotein contains < 1% of the total membrane-bound sialic acids and a very small fraction of the total SLex on neutrophil membranes. In spite of a relative resistance to sialidase digestion, the predominant form of sialic acid on the ligand is N-acetylneuraminic acid. Selective periodate oxidation of the side chain of sialic acids does not affect P-selectin binding and allows the introduction of tritium label into the truncated sialic acids. beta-Elimination with alkaline borohydride releases labeled O-linked oligosaccharides both from the labeled neutrophil ligand and from the ligand purified from HL-60 cells metabolically labeled with [3H]glucosamine. The ligand from both neutrophils and HL-60 cells is also susceptible to cleavage by the enzyme O-sialoglycoprotease from Pasteurella hemolytica. Analysis of the specificity of this enzyme suggests that the P-selectin ligand carries large numbers of closely spaced sialylated O-linked oligosaccharides. O-Sialoglycoprotease abolishes both direct binding of P-selectin to HL-60 cells and the adhesion of HL-60 cells to immobilized P-selectin, without significantly decreasing overall cell surface SLex expression. This indicates that the 120-kDa ligand may be the major determinant of P-selectin:myeloid cell interaction in vivo. Finally, based on the current and previous data, we hypothesize that the high affinity recognition site(s) of this P-selectin ligand may be derived from a "clustered saccharide patch" of sialylated fucosylated O-linked oligosaccharide sequences.