CD22/Siglec-2 is a B cell-specific molecule modulating surface IgM (sIgM) signaling via cytosolic tyrosine-based motifs. CD22 recognizes alpha2-6-linked sialic acids (Sias) via an amino-terminal Ig-like domain. This Sia-binding site is typically masked by unknown sialylated ligands on the same cell surface, an interaction required for optimal signaling function. We studied the effect of cell surface Sias on specific interactions of CD22 with other molecules and on its turnover via endocytosis. A novel approach for simultaneous biotinylation and cross-linking showed that CD22 associates with CD45 and sIgM at much higher levels than reported in prior studies, possibly involving cell surface multimers of CD22. Sia removal or mutation of a CD22 arginine residue required for Sia recognition did not affect these associations even in human:mouse heterologous systems, indicating that they are primarily determined by evolutionarily conserved protein-protein interactions. Thus masking of the Sia-binding site of CD22 involves many cell surface sialoglycoproteins, without requiring specific ligand(s) and/or is mediated by secondary interactions with Sias on CD45 and sIgM. Abrogating Sia interactions also does not affect constitutive CD22 endocytosis. Sia removal does enhance the much faster rate of anti-CD22 antibody-triggered endocytosis, as well as killing by an anti-CD22 immunotoxin. In contrast to the unstimulated state, sIgM cross-linking inhibits both antibody-induced endocytosis and immunotoxin killing. Thus the signal- modulating activity of CD22 Sia recognition cannot be explained by mediation of primary interactions with specific molecules, nor by effects on constitutive endocytosis. The effects on antibody-mediated endocytosis could be of relevance to immunotoxin treatment of lymphomas.