We evaluated the BacT/ALERT 3D system for recovery and drug susceptibility testing (DST) of Mycobacterium tuberculosis (MTB). Of 2659 clinical specimens, MTB was detected in 92 using BacT/ALERT, compared to 94 using Löwenstein-Jensen culture. Detection time was 25% shorter with BacT/ALERT. Sensitivities were 92%, 96%, 78% and 100% for resistance to rifampicin, isoniazid, streptomycin and ethambutol, respectively, while specificity was 100% for all antibiotics, when BacT/ALERT was compared with the BACTEC 460 method on 50 MTB isolates. The BacT/ALERT system is fully automated and creates no radioactive waste. It seems to be a valid alternative for primary isolation, but further evaluation is needed regarding DST.