The ability of a polymerase-chain-reaction-based technique combined with a reverse hybridisation line-probe assay to detect and identify eight of the most clinically significant mycobacterial species directly from cultures in liquid medium was evaluated. The line-probe assay allows simultaneous identification of eight different mycobacterial species. Ninety-seven mycobacterial strains belonging to 22 different species were evaluated. All strains were previously inoculated into MB/BacT bottles (Organon Teknika, USA). The sensitivity and specificity of the test when applied on positive MB/BacT liquid cultures containing isolates from mycobacterial species included as specific probes on the line-probe assay strip was 100% and 100%, respectively. These results further support the potential clinical usefulness of this technique in the diagnosis of mycobacterial infections.