Clostridium (C.) difficile, or its cytoxin, was demonstrated in faecal samples from 10 of 25 (40%) mature horses investigated with acute colitis treated primarily with antibiotics for disorders other than diarrhoea. C. difficile was not found in faecal samples from 140 horses without signs of enteric disorders, from 21 nondiarrhoeic horses treated with antibiotics, nor from 22 horses with colitis untreated with antibiotics. Except for C. difficile neither Salmonella nor any other investigated intestinal pathogen was isolated in any of the diarrhoeic horses. The findings strongly support some earlier reports that C. difficile is associated with acute colitis in mature horses treated with antibiotics. Of the 10 horses, 4 proved positive for C. difficile both in culture and in the cytotoxin test, 4 in culture only and 2 only in the cytotoxin test. Eight of 10 horses positive for C. difficile were or had recently been hospitalised, indicating that C. difficile may be a nosocomial infection in horses. All horses positive for C. difficile were treated with beta-lactam antibiotics.