To evaluate an interferon (IFN)-gamma release assay in diagnosing latent tuberculosis infection in pregnant adolescents and women at risk for exposure to Mycobacterium tuberculosis. This was a prospective study of women and adolescents receiving health care at Bellevue Hospital Outpatient Clinics in New York City. Each patient was assessed for M tuberculosis risk factors, had a tuberculin skin test placed, and an IFN-gamma release assay performed. The concordance between the tuberculin skin test and the IFN-gamma release assay was calculated and the results analyzed according to the likelihood of exposure to M tuberculosis. Mean mitogen IFN-γ levels were used across groups to compare reliability between trimesters and assay performance in pregnant compared with nonpregnant females of childbearing age. A total of 140 pregnant and 140 nonpregnant females were enrolled in the study. The IFN-gamma release assay was highly specific, and IFN-gamma release assay positivity was associated with a greater likelihood of exposure to M tuberculosis. The overall agreement between the tuberculin skin test and IFN-gamma release assay results was 88% for all pregnant patients, corresponding to a κ of 0.452 (confidence interval 0.26-0.64). Interferon-γ release from the mitogen did not appear to have any temporal association with pregnancy trimester in cross-sectional or longitudinal studies. The IFN-gamma release assay performed equally well in pregnant and nonpregnant females. The IFN-gamma release assay performed equally well in each trimester of pregnancy with comparable results to nonpregnant females. Interferon-gamma release assays are much more specific, at least as sensitive, and may be a better predictor of disease progression than the tuberculin skin test. : II.