By using dot blot hybridization, 69 of 102 Clostridium difficile isolates (68%) from the United States and other countries hybridized with at least one of nine DNA probes for erythromycin (Erm), tetracycline (Tet) or chloramphenicol (Cat) resistance determinants. The distribution of individual determinants in descending order of frequency was: Tet M, 32%; Erm Q, 25%; Erm FS, 18%; Tet P, 15%; Tet K, 15%; Cat P, 15%; Cat Q, 12%; Erm BP, 11%; Tet L, 7%. This is the first report of Tet P being carried by C. difficile and hitherto Erm FS has only been found within the genus Bacteriodes, while neither Tet K nor Tet L have been previously identified among the genus Clostridia. Eighteen percent of the hybridizing isolates carried multiple determinants coding for the same phenotype. A higher frequency of resistance genes was associated with prior exposure to antimicrobial agents, cytotoxin production and diarrhoea. Isolates recovered from bone marrow transplant patients carried significantly fewer antibiotic resistance genes than did those from immunocompetent general medicine patients. However, this may be due to the fact that each was located at a different site. Antibiotic resistance determinants may play a role in the virulence associated with C. difficile.