Clostridium difficile is the causative agent of a range of intestinal diseases, collectively referred to as Clostridium difficile -associated disease (CDAD). The recent emergence of “hypervirulent” strains associated with increased rates of mortality and severity of disease in humans has highlighted the need to study this organism at the molecular level. These studies will increase our knowledge of the mechanisms by which C. difficile causes disease and facilitate the rational design of new and improved therapeutics. The study of C. difficile has long been hampered by difficulties in genetically manipulating the organism. It has been only recently (within the last decade) that methods have been developed to introduce plasmid DNA into C. difficile and most importantly to enable the generation of isogenic mutants in this emerging human pathogen. These methods are essential prerequisites for the effective study of gene function in this important bacterium.