The new BDProbeTec ET System (BDET; BD Biosciences, Sparks, MD) was compared with the Roche COBAS AMPLICOR System (CAS) and culture for Mycobacterium tuberculosis (MTB). A total of 253 specimens (152 respiratory and 101 pleural fluid specimens) collected from 240 patients were tested in parallel with the 3 assays. After resolving the discrepancies, the sensitivity, specificity, and positive and negative predictive values of the BDET for detecting MTB was 76.9%, 93.7%, 71.4%, and 95.2% for the respiratory specimens and 88.9%, 92.4%, 53.3%, and 98.8% for the pleural fluid specimens, respectively. The corresponding values of the CAS were 69.2%, 100%, 100%, and 94% for the respiratory specimens and 33.3%, 100%, 100%, and 93.9% for the pleural fluid specimens, respectively. No significant differences in sensitivities were observed between the results of both assays for the respiratory specimens. However, statistically significant differences in sensitivities were found between the BDET and CAS for the pleural fluid specimens (P =.02). Although the BDET was less specific than the CAS (P =.007), the BDET has an excellent sensitivity for detecting MTB in the pleural fluid specimens. Considering the low sensitivity of other available tests, the BDET can be a useful diagnostic tool for excluding MTB, particularly in the pleural fluid specimens.