It is often not possible to obtain satisfactory sputum samples from patients suspected of having pulmonary tuberculosis. In this study, we asked whether it was possible to identify M. tuberculosis ribosomal RNA (rRNA) in mouthwash samples, and in a prospective clinical study, whether the presence of this rRNA correlated with clinical M. tuberculosis infection. Using a combination of reverse transcriptase and polymerase chain reaction amplification, it was possible to identify M. tuberculosis rRNA in mouthwash samples. M. tuberculosis rRNA was identified in mouthwash samples from patients with active tuberculosis more commonly than in samples from control subjects (P < 0.05). The test was positive in four of ten patients with active pulmonary tuberculosis, one of eight contacts, none of eight past cases of tuberculosis, two of eight patients with other diagnosis and one of five healthy volunteers. M. tuberculosis rRNA was identified in sputum from four of eight patients and in bronchoscopy trap samples from four of five patients with active tuberculosis. However, one of ten sputum samples and two of five bronchoscopy samples from subjects with a clinical diagnosis other than active tuberculosis were positive. These results indicate that although it is technically possible to identify M. tuberculosis on the basis of the presence of rRNA in mouthwash samples, the poor sensitivity and specificity of the technique suggest that it is unlikely to be useful clinically.