An environmental survey of a veterinary teaching hospital for the presence of Clostridium difficile was performed using contact plates and cycloserine-cefoxitin-fructose with 0.1% sodium taurocholate agar. Clostridium difficile was isolated from 24 of 381 sites (6.3%). Growth was obtained from 4.5% (9/202) of sites sampled in the Large Animal Clinic, from 8.1% (13/160) of sites within the Small Animal Clinic, and from 20% (2/10) of sites sampled elsewhere. Fourteen of 21 strains tested produced toxins in vitro. A geographic association was found with areas in the large animal clinic where nosocomial C. difficile diarrhea in horses had previously been diagnosed. Several other sites with a potential for nosocomial transmission of the organism were identified. Areas from which C. difficile was isolated tended to be areas with high animal traffic, with increased chance of fecal contamination, and with rough, difficult to clean surfaces. This study documents the prevalence of this organism in the environment and its potential role in nosocomial disease.