Toxigenic Clostridium difficile CCUG19126 produces the autoinducer-2 (AI-2) quorum sensing molecule that induces bioluminescence in Vibrio harveyi BBI70 reporter strain. AI-2-containing cell-free supernatants from mid-log phase C difficile and Escherichia coli DH5 alpha expressing recombinant luxS(cd) upregulated the transcript levels of tcdA (7-10-fold), tcdB (4-6-fold), and tcdE (2-3-fold) in early-log C. difficile. In contrast, no induction occurred when cells were exposed to sterile medium or to cell-free supernatant from E. coli DH5 alpha. AI-2 did not significantly increase the level of toxin A in early-log C. difficile. These results suggest that LuxS(cd)-dependent signaling regulates virulence gene expression at the transcriptional level in C. difficile. (C) 2005 Elsevier Inc. All rights reserved.