Cytochrome P450 CYP141 is an intermediary metabolic and respiratory protein that interferes with oxidation reduction in Mycobacterium tuberculosis. This conserved protein has also been debated as a hypothetical target for therapeutics. We used the sequences of CYP141 gene to develop a PCR for rapid detection of Mycobacterium tuberculosis from respiratory specimens. The sensitivity of this PCR for culture positive-smear positive and culture positive-smear negative samples were 92% and 62.5%, respectively. The overall sensitivity and specificity of this PCR was 85.7% and 97.8%. As compared with other studies, it appears that the CYP141 gene is a good target for direct detection of M. tuberculosis from respiratory specimens.