The cholera toxin B subunit (CTB) recognizes ganglioside GM1 receptors on target cells to facilitate entry of the toxin's A1 polypeptide into the host cytoplasm. GM1 binding to the CTB homopentamer occurs cooperatively with the most prominent interactions involving the terminal galactose residue of the ganglioside. Here, it is shown that association of galactose, lactose, or fucose (6-deoxy-galactose) with CTB is readily monitored using fluorescence spectroscopy. In many respects, however, the formation of CTB complexes with these small sugar analogues of GM1 greatly differs from the formation of complexes with the ganglioside itself. Each of these monosaccharides has a much weaker affinity for CTB than does GM1 and none of the sugars appear to be bound cooperatively. Moreover, GM1 binding conveys a stabilizing effect to CTB which is not seen upon binding of galactose or lactose. These data indicate that CTB-GM1 interactions involving sites other than the terminal galactose of the ganglioside serve prominently in the proper placement of CT on the target cell surface.