Our previous study (22) showed that a mixture of CT holotoxins produced in vivo and having chimeric B pentamers with from zero to two wild-type (wt) GM 1 binding sites (BS) was still capable of intoxicating host cells. Because genetic methods, not chemical modifications, were used to prepare these toxin variants, the structures of their GM 1 BS were fully defined and were known to be either the same as wt sites or inactive. Thus, having at most 2 wt GM 1 BS was sufficient for CT to intoxicate host cells, albeit at reduced efficiency. Left unanswered, however, was the question of whether a holotoxin with only one binding site for the GM 1 receptor can function. A toxin molecule able to bind only a single GM 1 molecule would be completely unable to cluster GM 1 molecules or scaffold them into microdomains and would therefore be unable to induce membrane curvature (2, 23) . We designed the experiments reported here to produce defined holotoxin variants that have from zero to five native GM 1 BS in their B pentamers, and we compared their abilities to bind to GM 1 and intoxicate T84 cells. We found that holotoxins able to interact with only a single GM 1 molecule can nevertheless still complete the intoxication process, demonstrating directly that binding a single GM 1 molecule permits the toxin to enter the host cell, complete the trafficking process, and deliver the toxic CT-A1 fragment to the cytoplasm. However, we also found that eliminating even a single binding site from wt holotoxin produced a detectable attenuation in toxicity.