Clostridium difficile is an emerging nosocomial pathogen and one of the major causes of antibiotic-associated diarrhoea. Cases of Clostridium difficile-associated disease (CDAD) are likely initiated by the ingestion of dormant C. difficile spores, which then germinate, outgrow and rapidly proliferate to cause gastrointestinal (GI) infections. To understand the initial stages of CDAD pathogenesis, we have characterized the germination of spores from a collection of C. difficile strains, including some clinical isolates obtained from a CDAD outbreak (CDAD isolates). Spores of one laboratory strain and five CDAD isolates did not germinate with amino acids, but did germinate on a nutrient-rich medium. However, bile salts had little effect on spore germination, either alone or in a nutrient-rich medium. These spores also germinated with KCl, as well as the non-nutrient germinants dodecylamine and a 1 : 1 chelate of Ca(2+) and dipicolinic acid. An unexpected finding was that spores of most of the C. difficile strains also germinated with inorganic phosphate (P(i)) with a pH optimum of 6. The in vitro germination of spores of CDAD strains with KCl and P(i), two molecules present at significant levels in the GI tract, suggests that C. difficile spores germinate in the human body by sensing P(i) in the early segments of the duodenum and KCl in the colon.