OBJECTIVE: Healthcare workers (HCWs), especially those caring for patients with tuberculosis (TB), are at high risk of acquiring that disease. The poor specificity of tuberculin skin testing (TST) prompted us to evaluate the effectiveness of the interferon-γ release assay (IGRA) in comparison with TST in a large prospective, multicenter, 1-year study of HCWs with occupational exposure to TB. METHODS: HCWs from high-risk units at 14 university hospitals were invited to participate and underwent both TST and IGRA (first Quantiferon TB Gold-IT®, QFT-G, then T-SPOT.TB® if QFT-G was indeterminate) at baseline and after 1 year. We collected demographic characteristics, country of birth, history of TB, immunosuppression, past exposure to TB, history of BCG vaccination, results of most recent TST, job category, and duration of current function. RESULTS: Among 807 HCWs enrolled, current or past TST at baseline was positive (≥15 mm) in 282 (34.9%); the IGRA was positive in 113 (14.0%) and indeterminate in 3 (0.4%). After 1 year, 594 HCWs had both an IGRA and TST (or prior TST≥15 mm) at baseline and an IGRA and TST (if indicated). The conversion rate was 2.5% (9 of 367) with TST and 7.6% (45 of 594) with IGRA, with poor agreement between the 2 tests. Using only QFT-G, conversion (9.9%) and reversion (17.8%) rates were higher for baseline QFT-G positive quantitative values <1 IU/mL. CONCLUSION: TST and the IGRA yielded discordant results. The value of IGRA in addition to TST remains undetermined; the two should be jointly interpreted in decision-making (clinical trial registration NCT00797836).