CD22 beta is a B cell surface glycoprotein involved in cell adhesion and activation. We previously reported that a recombinant soluble form termed CD22 beta Rg is capable of binding alpha 2-6 sialylated complex N-linked oligosaccharides purified from lymphocyte glycoprotein ligands (Powell, L. D., Sgroi, D., Sjoberg, E. R., Stamenkovic, I., and Varki, A. (1993) J. Biol. Chem. 268, 7019-7027). Here, we utilize a number of naturally and enzymatically sialylated oligosaccharides and sialoglycoproteins to further define its lectin specificity and demonstrate that the minimal structure recognized is Neu5Ac alpha 2-6Gal beta 1-4Glc(NAc). Reduction of the glucose residue of Neu5-Ac alpha 2-6Gal beta 1-4Glc diminishes the interaction, while truncation of the sialic acid side chain by mild periodate oxidation abolishes it. Branched oligosaccharides with two alpha 2-6-sialyl residues bind better, regardless of whether they were derived from N- or O-linked oligosaccharides or from gangliosides. alpha 2-3-Sialyl residues have no effect on binding, whereas increasing the number of alpha 2-6-sialyl residues on multiantennary oligosaccharides progressively improves binding. No specific feature of the core region affects binding, although the spacing of the alpha 2-6-sialyl residues on tetraantennary chains appears to have a significant effect. Of several model sialoglycoproteins examined, fetuin and transferrin had an apparent affinity no greater than that observed with free sialylated N-linked oligosaccharides. Some subfractions of these proteins displayed unexpectedly weak binding, suggesting that the protein backbone can exert a negative effect. In contrast, a subfraction of alpha 1-acid glycoprotein was identified as having a substantially higher apparent affinity than free oligosaccharides derived from it, indicating that multiple glycosylation sites may increase the apparent binding affinity. Thus, CD22 beta Rg contains a lectin activity specific for the minimal motif Neu5Ac alpha 2-6Gal beta 1-4Glc(NAc), and branched, multisialylated oligosaccharides are better ligands, regardless of the core sequences. Intact sialoglycoproteins can also interact, although with a variable affinity not directly predictable from the precise structure of their sialylated oligosaccharides chains. These data may help to explain why certain T and B cell surface sialoglycoproteins with the Neu5Ac alpha 2-6Gal beta 1-4Glc(NAc) motif are superior ligands, capable of mediating CD22 beta-mediated adhesion and activation events.