Clostridium difficile is the major causative agent of nosocomial antibiotic-associated diarrhea, colitis, and pseudomembranous colitis. The pathogenicity of C. difficile is closely related to the production of toxins A and B. Toxigenic C. difficile detection by a tissue culture cytotoxin assay is often considered the “gold standard.” However, this assay is time consuming, as it implies an incubation period of at least 24 h. We have developed a rapid real-time fluorescence-based multiplex PCR assay targeting the C. difficile toxin genes tcdA and tcdB, with the Smart Cycler. Two molecular beacons bearing different fluorophores were used as internal probes specific for each amplicon type. The analytical sensitivity of the assay was around 10 genome copies for all nine C. difficile strains tested, representing the 6 most common toxinotypes. The specificity was demonstrated by the absence of amplification with DNA purified from …