The aim of this study was to use restriction fragment length polymorphism to detect unsuspected cases of nosocomial transmission of tuberculosis (TB) among patients who had been admitted to a university hospital. One hundred fifty-one samples of Mycobacterium tuberculosis isolated from patients with pulmonary TB were studied. The isolates from 37 patients (24.5%) defined 11 clusters. None of the patients infected with these cluster isolates had hospital stays that coincided with one another, and for 5.4% of the patients, the epidemiological link was clearly outside the hospital. Previous incarceration was associated with infection with cluster isolates. In addition, 109 patients without TB (41 of whom were infected with human immunodeficiency virus) who shared a room with patients who had TB were followed for 18-60 months. Among the patients who survived, secondary cases of TB due to nosocomial transmission were not detected.