The aim of the present study has been to investigate the binding specificity of the soluble 14,000-dalton lectin of bovine heart muscle towards immobilised oligosaccharides in clustered form. To this end, chromatogram overlay assays and quantitative plastic-microwell-binding assays have been performed using several natural glycolipids and neoglycolipids containing one or more of the disaccharide units, beta-D-Galp-(1----4 or 3)-D-GlcNAc or beta-D-Galp-(1----4)-D-Glc and related structures. The microwell assay gave the most consistent results. It was observed that for binding by the soluble lectin the optimal sequence, which is beta-D-Galp-(1----4 or 3)-D-GlcNAc, must occur at the nonreducing end of longer oligosaccharides when linked to lipid. These oligosaccharides may be of poly(N-acetyllactosamine) type or they may be mono- or multi-antennary, complex-type chains in which the disaccharide is joined directly to a trimannosyl core. The lectin bound to such immobilised lipid-linked oligosaccharides on which the terminal D-galactosyl groups are substituted with alpha-L-Fucp-(1----2), alpha-D-Galp-(1----3), or alpha-NeuAc-(2----3) groups. However, no binding was detected if the terminal D-galactosyl groups were substituted with an alpha-NeuAc-(2----6) group or the subterminal N-acetylglucosamine units with an alpha-L-Fucp-(1----3 or -4) group. Internally located N-acetyllactosamine units where the D-galactose units are disubstituted by beta-D-GlcNacp-(1----3) and -(1----6) units, as in branched poly(N-acetyllactosamine) backbones were not bound by the bovine lectin. These results are in accord with previous observations on the bovine lectin and the corresponding human and rat lectins, using structurally defined oligosaccharides as inhibitors of binding. The results of comparative binding experiments using paragloboside and ceramide hexasaccharide which contain one and two N-acetyllactosamine units, respectively, joined in linear sequence to the lactosylceramide core, were equivocal with respect to the availability of the internal N-acetyllactosamine units for binding by the bovine lectin.