Clostridium difficile is a spore-forming bacterium that causes Clostridium difficile-associated disease (CDAD). Intestinal microflora keeps C. difficile in the spore state and prevents colonization. Following antimicrobial treatment, the microflora is disrupted, and C. difficile spores germinate in the intestines. The resulting vegetative cells are believed to fill empty niches left by the depleted microbial community and establish infection. Thus, germination of C. difficile spores is the first required step in CDAD. Interestingly, C. difficile genes encode most known spore-specific protein necessary for germination, except for germination (Ger) receptors. Even though C. difficile Ger receptors have not been identified, taurocholate (a bile salt) and glycine (an amino acid) have been shown to be required for spore germination. Furthermore, chenodeoxycholate, another bile salt, can inhibit taurocholate-induced C. difficile spore germination. In the present study, we examined C. difficile spore germination kinetics to determine whether taurocholate acts as a specific germinant that activates unknown germination receptors or acts nonspecifically by disrupting spores' membranes. Kinetic analysis of C. difficile spore germination suggested the presence of distinct receptors for taurocholate and glycine. Furthermore, taurocholate, glycine, and chenodeoxycholate seem to bind to C. difficile spores through a complex mechanism, where both receptor homo- and heterocomplexes are formed. The kinetic data also point to an ordered sequential progression of binding where taurocholate must be recognized first before detection of glycine can take place. Finally, comparing calculated kinetic parameters with intestinal concentrations of the two germinants suggests a mechanism for the preferential germination of C. difficile spores in antibiotic-treated individuals.