The frequency of introduction and spread of specific Clostridium difficile strains among hospitalized patients were assessed by serial cultures of patients admitted to a medical-surgical ward with endemic C. difficile-associated diarrhea. Stool cultures were obtained from 634 (94%) of 678 consecutive admissions to the ward (ward admissions), and all C. difficile isolates were typed by restriction endonuclease analysis. Sixty-five ward admissions introduced C. difficile to the ward, and 54 initially culture-negative admissions acquired C. difficile on the ward. Ward admissions hospitalized within the prior 30 days in the medical center were more likely to be culture-positive for C. difficile at admission to the study ward than those not previously hospitalized at the institution (16% vs. 7%, P less than .001). Nosocomial acquisition of a C. difficile strain was preceded by a documented introduction of that strain to the ward by another asymptomatic ward admission in 16 (84%) of 19 instances, suggesting that C. difficile-colonized new admissions are a major source of nosocomial C. difficile infections.