This research investigated the role of the anaerobic bacterium ' Clostridium difficile' in equine enterocolitis. The objectives of this study were to compare the prevalence of the isolation of 'Clostridium difficile' and detection of 'C. difficile' toxins A and/or B from diarrheic adult horses and foals versus those with normal feces. 'Clostridium difficile' was isolated from 7/55 diarrheic adult horses (13%) versus 1/255 adult horses with normal feces (0.4%) (p < 0.001). 'Clostridium difficile' toxins were present in the feces of 12/55 diarrheic adults (22%) versus 1/83 adults with normal feces (1.2%) (p < 0.001). Two culture positive/toxin negative and 7 culture negative/toxin positive cases were encountered. 'Clostridium difficile' was isolated from 11/31 diarrheic foals (35%) versus 0/40 foals with normal feces (p < 0.001). 'Clostridium difficile' toxins were detected in the feces 5/30 diarrheic foals (17%) and 0/21 normal foals (p < 0.05). Fecal samples from 8 foals were culture positive/toxin negative and 2 were culture negative/toxin positive. To clarify the significance of culture positive/toxin negative and culture negative/toxin positive cases, the survival of 'C. difficile' and persistence of 'C. difficile' toxins were studied. The recovery of 'C. difficile' in aerobically stored feces decreased dramatically over the first 72 hours at which point only 26% of samples yielded growth. This was determined to be due to poor aerotolerance because ' C. difficile' persisted in fecal samples stored anaerobically. In contrast, 'Clostridium difficile' toxins were determined to be immunologically stable in equine feces stored under aerobic conditions. 'Clostridium difficile' spores were isolated from 24/381 sites within the Ontario Veterinary College Veterinary Teaching Hospital including 9/202 (4.5%) sites in the large animal clinic, 13/160 (8.1%) sites in the small animal clinic and 2/10 (20%) sites in other areas. Forty-one/43 equine and environmental 'C. difficile' isolates were resistant to bacitracin and cefotaxime. All isolates were sensitive to vancomycin and metronidazole. Variable resistance was present against penicillin G, tetracycline, erythromycin and trimethoprim-sulfa. This study provides further information into the role of 'C. difficile ' in sporadic and nosocomial enterocolitis in adult horses and foals. However, a great deal of future study is required to better understand the role of this pathogen in equine disease.