Glycan recognition by lectins initiates clinically relevant processes such as toxin binding or tumor spread. Thus, the development of potent inhibitors has a medical perspective. Toward this goal, we report the synthesis of both rigid and flexible bivalent lactosides on scaffolds that include secondary and tertiary terephthalamides and N,N0-diglucosylterephthalamides. Construction of these compounds involved Schmidt-Michel glycosidation, and amide coupling or Ugi reactions of relevant glycosyl amines in key steps. A glycocluster based on a rigid glycophane was also prepared from coupling of a glucuronic acid derivative and p-xylylenediamine with subsequent ring-closing metathesis. Finally, a more flexible bivalent lactoside was produced from lactosyl azide with use of the copper-catalyzed azide-alkyne cycloaddition. Distances between lactose residues were analyzed computationally as were their orientations for the relatively rigid subset of compounds. Distinct spacing properties were revealed by varying the structure of the scaffold or by varying the location of the lactose residue on the scaffold. To relate these features to bioactivity a plant toxin and human adhesion/growth-regulatory galectins were used as sensors in three types of assay, i.e. measuring agglutination of erythrocytes, binding to glycans of a surface-immobilized glycoprotein, or binding to human cells.Methodologically, the common hemeagglutination assaywas found to be considerably less sensitive than both solid-phase and cell assays. The bivalent compounds were less effective at interfering with glycoprotein binding to the plant toxin than to human lectins. Significantly, a constrained compound was identified that displayed selectivity in its inhibitory potency between galectin-3 and its proteolytically processed form. Conversely, compounds with a high degree of flexibility showed notable ability to protect human cells fromplant toxin binding. The applied conjugation chemistry thus is compatiblewith the long-termaim to produce potent and selective lectin inhibitors.