We have isolated and purified enolase from Clostridium difficile. This is the first report of an enolase of the Clostridium genus, and in general its characteristics resemble those described previously for other species, except that it is extremely thermostable. Interestingly, C. difficile enolase has an octameric structure (approximately 300 kDa on native PAGE, 50 kDa on SDS PAGE, and 338 kDa by gel filtration). Enolases from C. sordellii and C. bifermentans have been partially purified and have a molecular weight similar to that of C. difficile. It may be that this large size is common for enolases isolated from bacteria of the Clostridium genus.