Patients hospitalized in a hospital with a high incidence of antibiotic-associated diarrhea due to toxin A-negative, toxin B-positive (A-/B+) Clostridium difficile were retrospectively investigated to determine the clinical manifestations and risk factors for infection. Of 77 Clostridium difficile isolates obtained from 77 patients during the 1-year investigation period, 30 were A-/B+ and 47 were toxin A-positive, toxin B-positive (A+/B+). By pulsed-field gel electrophoresis analysis, 23 of the 30 A-/B+ strains were outbreak-related, suggesting nosocomial spread of a single type of bacterium, which mainly affected patients in the wards of respiratory medicine, hematology and neurology. Using regression analysis, three factors were found to be associated with infection by A-/B+ isolates: (i) exposure to antineoplastic agents ( P=0.01, odds ratio [OR]=5.1), (ii) the use of nasal feeding tubes ( P=0.008, OR=5.2), and (iii) assignment to a certain internal medicine ward ( P=0.05, OR=3.0). Between patients with Clostridium difficile-associated diarrhea caused by A-/B+ strains and those with A+/B+ strains, no statistically significant difference was found in body temperature, serum concentration of C-reactive protein, leukocyte count in whole blood, frequency of diarrhea, or type of underlying disease. These results indicate that A-/B+ strains of Clostridium difficile can cause intestinal infection in humans and they spread nosocomially in the same manner as A+/B+ strains.