In previous studies, we showed that diet composition or Saccharomyces boulardii ingestion could protect gnotobiotic mice against lethal Clostridium difficile infection. Using an original method, we detected nontoxinogenic clones from feces of protected mice challenged with a toxinogenic clone of C. difficile. These clones became established at the same level as the toxinogenic one after about 30 days. In these protected mice bearing nontoxinogenic clones, no enterotoxin production could be detected and cytotoxin titers were highly reduced. These nontoxinogenic clones were genetically stable because nontoxinogenic clones and clones that produce intermediate levels of toxins in vivo did not revert to toxin production, even after repeated culture in vitro. Furthermore, the nontoxinogenic clones were shown to arise from a single toxinogenic clone and were identical to that clone in metabolic patterns and antibiotic sensitivity tests. When mice fed a nonprotective diet were challenged with a nontoxinogenic or intermediate clone, they remained healthy and no toxin production could be detected in their feces. Moreover, these mice were protected against further infections with toxinogenic strains of C. difficile, and a strong antagonism between nontoxinogenic and toxinogenic clones was observed.