The study was designed to estimate prevalence of tuberculosis infection among health care workers, using the tuberculin skin test (TST) and the new M. tuberculosis specific diagnostic whole-blood test and to identify possible risk factors. Employees at 2 departments of infectious diseases in Copenhagen were invited to enter the study. All attendants completed a questionnaire, had a TST and blood drawn for detection of interferon-gamma produced after stimulation with M. tuberculosis specific antigens ESAT-6 and CFP-10 (QuantiFERON-TB-Gold, Cellestis). 47 of 139 (34%) participants had a positive TST whereas only 2 of 139 (1%) had a positive QuantiFERON TB-Gold test (QFT-TB). 42 of 106 (40%) BCG vaccinated had positive TST (> or =12 mm) compared with 2 of 27 (7%) unvaccinated persons. Among 47 persons with positive TST, 42 (89%) were BCG- vaccinated. The 2 QFT-TB positive participants as well as the remaining 45 TST positive participants showed no sign of active tuberculous disease and were allocated to 6-month clinical follow-up, without medical therapy. Today, 1.5 y later, all remain healthy. The high rate of positive TST among health care workers was most probably due to BCG vaccination and not to infection with M. tuberculosis. The overall transmission rate determined by QFT-TB was found to be very low. The QFT-TB may be useful in distinguishing persons with latent TB infection from persons with positive TST due to BCG vaccination and its use may reduce anxiety.