A nurse working in a newborn nursery and maternity ward developed 3+ smear-positive lung tuberculosis. The hospital infection control committee, in collaboration with the local public health and welfare center, conducted a contact investigation. The infection period was defined as April to August 2006. The investigation included 109 infant and mother pairs, 28 children aged under 10 years and their guardians, 62 coworkers, and 63 household visitors to the ward. Tuberculosis infection in infants and children aged under 5 years was primarily determined by tuberculin skin test (TST), while subjects aged 5 years or more were tested using QuantiFERON-TB Gold (QFT). The first investigation, in August 2006, was conducted in all subjects, and the second investigation, in October 2006, targeted selected subjects. No infants were TST-positive. Two children aged 1 year or under, vaccinated with bacillus Calmette-Guérin, were positive for TST, as determined by the criteria of the Japan Anti-Tuberculosis Association; however, other tests for tuberculosis were negative. Of the 13 QFT-positive adult subjects, 1 mother and 2 coworkers could have become infected with Mycobacterium tuberculosis through exposure to the index nurse. Fifty-four infants and 6 children underwent "window-period" prophylaxis, and 4 adults completed 6-month prophylactic treatment with isoniazid. To date, no secondary cases of tuberculosis disease have occurred.