The major controversy in the diagnosis of symptomatic gastrointestinal infection due to Clostridium difficile is whether laboratory evidence of the C. difficile organism in culture is sufficient or if evidence of one of the C. difficile toxins in stool should be required. Cultures performed properly on selective media currently are the most sensitive method for detection of C. difficile, whereas the cell cytotoxin assay for detection of toxin B is the most specific. Stool specimens from patients with clinical diarrhea are sometimes found to be culture-positive for C. difficile but assay-negative for cytotoxin. Samples from these patients can be viewed as false-positive by culture or false-negative by cytotoxin test. Evidence from endoscopy indicates that some patients whose stool is culture-positive for the organism but assay-negative for toxin do have pseudomembranous colitis, but its incidence among such patients (11%) is lower than that among patients whose stool is culture- and assay-positive (51%). Response to treatment with vancomycin or metronidazole is similar in the two groups of patients, and withholding treatment from patients whose stool contains C. difficile but not cytotoxin may result in increased morbidity and mortality. Up to one-third of C. difficile organisms from stool specimens that are culture-positive but assay-negative are incapable of producing cytotoxin in vitro, a finding that suggests these organisms may not be the cause of diarrhea.(ABSTRACT TRUNCATED AT 250 WORDS)