Clostridium difficile is a Gram-positive sporulating anaerobic bacillus which causes pseudomembranous colitis. Nosocomial acquisition of this bacteria has proved frequent, and epidemiological markers are needed to recognize and control common-source outbreaks. We therefore compared the results of pulsed-field gel electrophoresis (PFGE) after restriction with SmaI or NruI, random-amplified polymorphic DNA (RAPD) using 3 10-mer oligonucleotides, and ribotyping to differentiate between 30 unrelated strains of C. difficile belonging to 8 serotypes. The strains were separated into 26 different types by PFGE, 25 by RAPD, but into only 18 types by ribotyping. Median percentages of similarity between strains ranged from 27 in the PFGE assay to 90 in the ribotyping assay, but there was good agreement between the 3 methods for the clustering of strains. PFGE was more time-consuming than RAPD but its patterns were easier to analyze.