This work shows that multivalent binding to GM 1 is indeed dispensable for CT toxicity. To do this, we prepared and purified homogenous preparations of holotoxins with defined numbers of GM 1 BS. Monomeric binding of CT to single molecules of GM 1 permits the holotoxin to intoxicate the host cell. Our results confirm and expand our studies on trafficking of the single GM 1 lipids. Conversely, loss of even a single GM 1 BS resulted in a measurable diminishment of toxicity in the T84 line of polarized human intestinal cells. This result is also consistent with findings of our studies of trafficking of the non-cross-linked GM 1 molecules, where we found that GM 1 cross-linking by toxin binding enhanced entry of CT into the ER. Thus, although multivalent binding to GM 1 by CT is fundamentally dispensable, it does have a significant effect on retrograde trafficking from the cell surface to the ER and on toxicity.