The pathogenicity locus (PaLoc) of Clostridium difficile contains toxin A and B genes and three accessory genes, including tcdD and tcdC, which are supposed to code for the positive and negative regulators of toxin expression, respectively. Different studies have described variations in C. difficile toxin A and B genes, but little is known about C. difficile variants for the accessory genes. The PaLoc of several C. difficile clinical isolates was investigated by three different PCR methods with the aim to identify variant strains. Of the toxinogenic C. difficile strains examined, 25% showed variations. No correlation between C. difficile variant strains and key patient groups was found. Interestingly, all of these strains showed a variant tcdC gene. Three different tcdC alleles were identified, and one of these had a nonsense mutation which reduced the TcdC protein from 232 to 61 amino acids. It is possible that different TcdC variants affect toxin production differently, a hypothesis with important implications for the pathogenic potential of variant C. difficile strains.