Rapid detection of resistance in Mycobacterium tuberculosis can optimize the efficacy of antituberculous therapy and control the transmission of resistant M. tuberculosis strains. Real-time PCR has minimized the time required to obtain the susceptibility pattern of M. tuberculosis strains, but little effort has been made to adapt this rapid technique to the direct detection of resistance from clinical samples. In this study, we adapted and evaluated a real-time PCR design for direct detection of resistance mutations in clinical respiratory samples. The real-time PCR was evaluated with (i) 11 clinical respiratory samples harboring bacilli resistant to isoniazid (INH) and/or rifampin (RIF), (ii) 10 culture-negative sputa spiked with a set of strains encoding 14 different resistance mutations in 10 independent codons, and (iii) 16 sputa harboring susceptible strains. The results obtained with this real-time PCR design completely agreed with DNA sequencing data. In all sputa harboring resistant M. tuberculosis strains, the mutation encoding resistance was successfully detected. No mutation was detected in any of the susceptible sputa. The test was applied only to smear-positive specimens and succeeded in detecting a bacterial load equivalent to 10(3) CFU/ml in sputum samples (10 acid-fast bacilli/line). The analytical specificity of this method was proved with a set of 14 different non-M. tuberculosis bacteria. This real-time PCR design is an adequate method for the specific and rapid detection of RIF and INH resistance in smear-positive clinical respiratory samples.