Cholera toxin (CT) moves from the plasma membrane (PM) of host cells to the endoplasmic reticulum (ER) by binding to the lipid raft ganglioside GM₁. The homopentomeric B-subunit of the toxin can bind up to five GM₁ molecules at once. Here, we examined the role of polyvalent binding of GM₁ in CT action by producing chimeric CTs that had B-subunits with only one or two normal binding pockets for GM₁. The chimeric toxins had attenuated affinity for binding to host cell PM, as expected. Nevertheless, like wild-type (wt) CT, the CT chimeras induced toxicity, fractionated with detergent-resistant membranes extracted from toxin-treated cells, displayed restricted diffusion in the plane of the PM in intact cells, and remained bound to GM₁ when they were immunoprecipitated. Thus, binding normally to two or perhaps only one GM₁ molecule is sufficient for association with lipid rafts in the PM and toxin action. The chimeric toxins, however, were much less potent than wt toxin, and they entered the cell by endocytosis more slowly, suggesting that clustering of GM₁ molecules by the B-subunit enhances the efficiency of toxin uptake and perhaps also trafficking to the ER.