The present studies explore multivalent ligand-receptor interactions between pentameric cholera toxin B subunits (CTB) and the corresponding membrane ligand, ganglioside GM(1). CTB binding was monitored on supported phospholipid bilayers coated on the walls and floors of microfluidic channels. Measurements were made by total internal reflection fluorescence microscopy (TIRFM). Apparent dissociation constants were extracted by fitting the binding data to both the Hill-Waud and Langmuir adsorption isotherm equations. Studies of the effect of ligand density on multivalent CTB-GM(1) interactions revealed that binding weakened with increasing GM(1) density from 0.02 mol % to 10.0 mol %. Such a result could be explained by the clustering of GM(1) on the supported phospholipid membranes, which in turn inhibited the binding of CTB. Atomic force microscopy (AFM) experiments directly verified GM(1) clustering within the supported POPC bilayers.