Oligosaccharide recognition by three mammalian mannose-binding proteins was investigated by using as probes a series of structurally characterized neoglycolipids in t.l.c. binding assays. The neoglycolipids were derived from N-linked oligosaccharides of complex, high-mannose and hybrid types and from human milk oligosaccharides and simple di- and tri-saccharides. The three proteins, namely the recombinant carbohydrate-recognition domain of rat mannose-binding Protein A and the multi-subunit forms of rat and human serum mannose-binding proteins, were shown to have in common reactivity with oligosaccharide probes containing one or more non-reducing terminal N-acetylglucosamine residue(s). Substitution with galactose masks reactivity. The three proteins also bound to non-reducing terminal mannose residues in high-mannose-type oligosaccharides, non-reducing terminal fucose residues in the sequence Fuc alpha 1-4(Gal beta 1-3)GlcNAc and non-reducing terminal glucose residues in dextran oligomers; the recombinant binding domain gave consistently weaker binding. The relative reactivities with the various probes differ for each protein. Overall, the reaction patterns of the three mammalian proteins differ from that of the plant lectin concanavalin A, which showed preferential binding to the high-mannose type, weak binding to biantennary complex type and no binding to the fuco-oligosaccharide and simple oligosaccharide probes. As a group, the three mammalian proteins resemble bovine serum conglutinin and behave as lectins with rather broad sugar specificities directed at certain non-reducing terminal N-acetylglucosamine, mannose, glucose and fucose residues, but with subtle differences in fine specificities. These results illustrate the potential of neoglycolipids in studies of oligosaccharide recognition by natural and recombinant proteins of diverse biological systems.