Despite empirical clinical association of infection with Clostridium difficile with colitis in horses, a causal link has not been confirmed. The objective of this study was to develop a model of C. difficile-associated diarrhea in foals with normal transfer of passive immunity. Nine 1-day-old pony foals were inoculated intragastrically with spores or vegetative cells of C. difficile. Five foals were challenged with spores, with 2 receiving 10(5) colony-forming units (CFUs) and concurrently 3 receiving 10(7) CFUs once daily for 3 days. Clindamycin was administered orally to disrupt gastrointestinal flora. A further 4 foals were challenged by orogastric administration of 10(10) CFUs of vegetative cells once daily for 3 days or until diarrhea developed. This group did not receive clindamycin. Spore and vegetative cell preparations were negative for toxins of C. difficile and common enteropathogens. Clinical signs varied from mild abdominal discomfort and pasty feces to colic and watery diarrhea in 8 of 9 foals. Four of 5 foals challenged with spores developed mild diarrhea, whereas all foals challenged with vegetative cells developed moderate to severe diarrhea. C. difficile was isolated from feces of all foals between 24 and 72 hours after inoculation and toxins A or B or both were detected in the feces of all foals by an enzyme-linked immunosorbent assay. We concluded that spores and vegetative cells of C. difficile are capable of colonizing the gastrointestinal tract, producing toxins, and inducing clinical signs similar to those encountered in naturally occurring cases. This study fulfilled Koch's postulates for C. difficile-associated diarrhea in foals and provides a model for consistent reproduction of the disease for future studies.