Binding of the N-acetyllactosamine-specific lectin from Erythrina corallodendron (ECorL) to four glycosphingolipids has been tested using the microtiter well assay. The role of several amino acids in the binding site region was studied by combining binding assays and molecular modeling for native and recombinant forms of the lectin. Seven single-point mutants at positions 106 (Y106A), 108 (Y108A, T), 218 (A218G), and 219 (Q219A, N or E) were investigated. A comparison with more than 30 known sequences of legume lectins showed that ECorL is unique in displaying a tyrosine residue or a structural equivalent at position 106. Analyses of the binding results obtained for mutants at positions 106 and 108 using molecular modeling point to complex conformational dependencies between these and several other residues around the binding site. Gln 219 was found to have a large conformational flexibility, which, paradoxically, favors the binding of N-acetyllactosamine-containing glycosphingolipids. Particularly significant is the fact that ECorL exhibits a higher affinity for Fuc alpha2Gal beta4GlcNAc beta-terminated glycosphingolipids than N-acetyllactosamine-terminated ones, in accordance with molecular modeling revealing a perfect fit of the alpha2-linked fucose in a cavity extending from the Gal beta4 binding pocket. These findings lead to a redefinition of the specificity of this lectin, where the affinity for the terminal Fuc alpha2Gal beta4GlcNAc beta trisaccharide should be considered in the first place. The possible biological significance of this specificity remains to be investigated.