Antibiotic-associated colitis (AAC) is confirmed by the isolation of Clostridium difficile cytotoxin from stool in patients with diarrhea. Culture of the organism has not been required to confirm the diagnosis. A review of cases of C. difficile culture-positive patients was performed in an attempt to clarify the significance of culture-positive toxin-negative (CPTN) compared to culture-positive toxin-positive (CPTP) disease. During an 11-month period, 45 patients were identified who had stool cultures positive for C. difficile. Sixteen of the patients studied were CPTP and 29 were CPTN. There were no major differences between the two groups for underlying diseases, antibiotic exposure, or diagnostic testing. Of the CPTP patients, 10 were treated for AAC and all responded. Two untreated patients resolved spontaneously. Of the CPTN patients, none was given specific antibiotic therapy, symptoms spontaneously resolved in 17, and symptoms were unresolved in five (colectomy or expired before resolution). A prospective analysis was performed of all C. difficile isolated from stool samples by the microbiology laboratory. Isolates were incubated in vitro and cytotoxin production was measured. Of isolates from CPTP patients 97% produced cytotoxin compared to 67% of isolates from CPTN patients (p less than 0.005). The results suggest that C. difficile, despite the absence of cytotoxin, may be an etiological factor in certain diarrheal syndromes. Until a randomized therapeutic trial for CPTN patients is conclusive, a positive culture should be considered evidence for treatment of patients with persistent diarrhea.