Twenty years ago, Clostridium difficile was first established as a cause of pseudomembranous colitis and antibiotic-associated diarrhea.C. difficile diarrhea is a widely recognized problem in the inpatient setting, with potentially significant morbidity and mortality. Antibiotics, and some chemotherapy agents, can potentially cause C. difficile colitis/diarrhea. The most commonly implicated agents are ampicillin, clindamycin, and cephalosporins. Diarrhea during antibiotic therapy is common and may be caused by C. difficile. Testing for C. difficile differentiates diarrheas into C. difficile positive and C. difficile negative. C. difficile can be carried asymptomatically as normal gastrointestinal flora, and in adults who have received antibiotic therapy, carrier states can be as high as 46%. Hospitalized patients are often colonized with C. difficile. C. difficile produces 3 virulence factors: an enterotoxin (toxin A), a cytotoxin (toxin B), and a substance to inhibit bowel motility. Different tests can be used to detect these toxins. The most widely used test is the enzyme immunoassay (EIA) for toxin A, toxin B, or both. The EIA C. difficile toxin assay has sensitivity and specificity ranges of 50% to 90% and 70% to 95%, respectively. Diagnostically, C. difficile cell culture cytotoxin assay remains the gold standard with sensitivity and specificity of 93% and 89%, respectively. Because of lack of confidence of the EIA for C. difficile, some clinicians assume an initial negative result may represent a false-negative test, and repeat testing is often done. We evaluated the value of repeat stool testing for C. difficile toxin A and B by EIA in inpatients with nosocomial diarrhea on antibiotics.