Previous studies suggested that 7 to 15% of healthy adults are colonized with toxigenic Clostridium difficile. To investigate the epidemiology, genetic diversity, and duration of C. difficile colonization in asymptomatic persons, we recruited healthy adults from the general population in Allegheny County, Pennsylvania. Participants provided epidemiological and dietary intake data and submitted stool specimens. The presence of C. difficile in stool specimens was determined by anaerobic culture. Stool specimens yielding C. difficile underwent nucleic acid testing of the tcdA gene segment with a commercial assay; tcdC genotyping was performed on C. difficile isolates. Subjects positive for C. difficile by toxigenic anaerobic culture were asked to submit additional specimens. One hundred six (81%) of 130 subjects submitted specimens, and 7 (6.6%) of those subjects were colonized with C. difficile. Seven distinct tcdC genotypes were observed among the 7 C. difficile-colonized individuals, including tcdC genotype 20, which has been found in uncooked ground pork in this region. Two (33%) out of 6 C. difficile-colonized subjects who submitted additional specimens tested positive for identical C. difficile strains on successive occasions, 1 month apart. The prevalence of C. difficile carriage in this healthy cohort is concordant with prior estimates. C. difficile-colonized individuals may be important reservoirs for C. difficile and may falsely test positive for infections due to C. difficile when evaluated for community- acquired diarrhea caused by other enteric pathogens.