Strain 8864 is a natural isolate of Clostridium difficile that is toxin B-positive and toxin A-negative. Recent work showed that there is a genetic rearrangement occurring at the pathogenicity locus (PaLoc) of the bacteria. Our investigation in the PaLoc region revealed an open reading frame (tcdF) of 543 bp DNA not reported before. This tcdF could encode a putative polypeptide of 22 kDa. Although no peptide homology was found with other known proteins, we postulate that it could be a novel protein because not only highly consensus ribosome-binding and promoter sequences were found upstream of tcdF, transcript was also identified at the region occupied by tcdF. ß