The performance was evaluated of a fluorescence in situ hybridisation assay using peptide nucleic acid probes (Dako Probe MTB Culture Confirmation Test; Dako, Denmark) for identification of Mycobacterium tuberculosis complex (MTC) organisms and differentiation between tuberculous and non-tuberculous mycobacteria (NTM) in material taken directly from Bactec 12B (Becton Dickinson, USA) and MB/BacT (Organon Teknika, USA) bottles. The test was applied to 129 smear-positive (Ziehl-Neelsen stain) clinical specimens, 48 previously identified clinical strains of mycobacteria (12 MTC and 36 NTM), and 51 reference strains (7 MTC and 44 NTM) which were all previously inoculated into Bactec 12B and MB/BacT bottles. The sensitivity and specificity of the assay for MTC-positive cultures was 87.6% and 100%, respectively, for Bactec 12B, and 100%, respectively, for MB/BacT. The sensitivity and specificity of the assay for NTM-positive cultures was 100% for both media.