The aim of this study was to compare the rate of recovery of mycobacteria and the time to detection in 5208 samples using the MB/BacT culture system (Organon Teknika, USA) and Löwenstein-Jensen medium. Mycobacteria were recovered from 301 (5.7%) samples. Two hundred fifty-seven (85.3%) isolates from 114 patients were Mycobacterium tuberculosis [135 (52.5%) smear-positive, 122 (47.4%) smear-negative], and 44 (14.6%) were potentially pathogenic environmental mycobacteria. The yield with the MB/BacT was higher than that with Löwenstein-Jensen [287 (95.3%) vs. 200 (66.4%), P<0.001] for both Mycobacterium tuberculosis [247 (96.1%) vs. 187 (72.7%), P<0.001] and potentially pathogenic environmental mycobacteria [40 (90.9%) vs. 13 (29.5%), P<0.001], mainly at the expense of the smear-negative samples. Moreover, 70 (27.2%) samples were positive only in the MB/BacT, whereas ten (3.8%) samples were positive only in Löwenstein-Jensen. The number of patients with tuberculosis detected by the MB/BacT was higher than that detected by Löwenstein-Jensen medium [111 (97.3%) vs. 89 (78%), P<0.001]. In 25 (21.9%) patients the diagnosis was established solely by means of the MB/BacT. In smear-positive and smear-negative samples, the mean times to detection of Mycobacterium tuberculosis were 16.7 and 26.3 days, respectively, with Löwenstein-Jensen and 11.5 and 19.3 days, respectively, with the MB/BacT. These results indicate that the MB/BacT is more efficient and faster than Löwenstein-Jensen for the recovery of mycobacteria.