Disseminated tuberculosis was diagnosed at the autopsy of a 65-day-old premature infant who died in a 52-bed neonatal intensive care unit (NICU). Both parents and one sibling had previously had positive tuberculin skin tests (TSTs); none had active pulmonary tuberculosis, but a second sibling had hilar adenopathy. Congenital transmission was confirmed by isolation of Mycobacterium tuberculosis from the mother's endometrium and the infant's lung tissue. Both strains were identical by DNA restriction fragment analysis. TSTs were performed on 14 neonates, 27 NICU visitors, 11 contacts of the family, and 260 health care workers. TST conversion occurred in two nurses (0.8%); both had normal chest radiographs and received isoniazid therapy. Exposed neonates had negative chest radiographs, had negative gastric aspirates for acid-fast bacilli, and received isoniazid preventive therapy. Diagnosis of congenital tuberculosis requires a high index of suspicion. Transmission of tuberculosis in the NICU setting is unusual but can occur.