We combined real-time RT-PCR and real-time PCR (R/P) assays using a hydrolysis probe to detect Mycobacterium tuberculosis complex (MTBC)-specific 16S rRNA and its rRNA gene (rDNA). The assay was applied to 28 nonrespiratory and 207 respiratory specimens from 218 patients. Total nucleic acids (including RNA and DNA) were extracted from samples, and results were considered positive if the repeat RT-PCR threshold cycle was < or =35 and the ratio of real-time RT-PCR and real-time PCR load was > or =1.51. The results were compared with those from existing methods, including smear, culture, and real-time PCR. Following resolution of the discrepant results between R/P assay and culture, the overall sensitivity, specificity, positive predictive values (PPV), and negative predictive values (NPV) of all samples (including nonrespiratory and respiratory specimens) were 98.2%, 97.2%, 91.7%, and 99.4%, respectively, for R/P assay, and 83.9%, 89.9%, 72.3%, and 94.7%, respectively, for real-time PCR. Furthermore, the R/P assay of four patient samples showed a higher ratio before treatment than after several days of treatment. We conclude that the R/P assay is a rapid and accurate method for direct detection of MTBC, which can distinguish viable and nonviable MTBC, and thus may guide patient therapy and public health decisions.