induces antibiotic-associated diarrhea due to the release of toxin A (TcdA) and toxin B (TcdB), the latter being its main virulence factor. The epidemic strain NAP1/027 has an increased virulence attributed to different factors. We compared cellular intoxication by TcdB with that by the reference strain VPI 10463 (TcdB). In a mouse ligated intestinal loop model, TcdB induced higher neutrophil recruitment, cytokine release, and epithelial damage than TcdB. Both toxins modified the same panel of small GTPases and exhibited similar in vitro autoprocessing kinetics. On the basis of sequence variations in the frizzled-binding domain (FBD), we reasoned that TcdB and TcdB might have different receptor specificities. To test this possibility, we used a TcdB from a NAP1 variant strain (TcdB) unable to glucosylate RhoA but with the same receptor-binding domains as TcdB. Cells were preincubated with TcdB to block cellular receptors, prior to intoxication with either TcdB or TcdB. Preincubation with TcdB blocked RhoA glucosylation by TcdB but not by TcdB, indicating that the toxins use different host factors for cell entry. This crucial difference might explain the increased biological activity of TcdB in the intestine, representing a contributing factor for the increased virulence of the NAP1/027 strain.