To investigate the influence of chemical and physical factors on the rate and extent of germination of Clostridium difficile spores. Germination of C. difficile spores following exposure to chemical and physical germinants was measured by loss of either heat or ethanol resistance. Sodium taurocholate and chenodeoxycholate initiated germination together with thioglycollate medium at concentrations of 0.1-100 mmol l(-1) and 10-100 mmol l(-1) respectively. Glycine (0.2% w/v) was a co-factor required for germination with sodium taurocholate. There was no significant difference in the rate of germination of C. difficile spores in aerobic and anaerobic conditions (P > 0.05) however, the initial rate of germination was significantly increased at 37 degrees C compared to 20 degrees C (P < 0.05). The optimum pH range for germination was 6.5-7.5, with a decreased rate and extent of germination occurring at pH 5.5 and 8.5. This study demonstrates that sodium taurocholate and chenodeoxycholate initiate germination of C. difficile spores and is concentration dependant. Temperature and pH influence the rate and extent of germination. This manuscript enhances the knowledge of the factors influencing the germination of C. difficile spores. This may be applied to the development of potential novel strategies for the prevention of C. difficile infection.