Rapid and sensitive tools for the diagnosis of tuberculosis are needed, due to the increased incidence of tuberculosis epidemics and the length of time required by classical diagnostic tests, especially among human immunodeficiency virus (HIV)-infected patients. In this context, the recent advances in cloning and characterization of M. tuberculosis genes has allowed the application of basic molecular biology techniques to the examination of clinical samples, such as sputum and bronchoalveolar lavage (BAL), for the molecular diagnosis of tuberculous infection. By using the polymerase chain reaction (PCR) for the amplification of mycobacterial nucleic acids and nonradiometric revelation techniques, the time required for the identification of mycobacteria has been considerably shortened (24-48 h), in comparison to the time required by microbiological tests. When PCR technique is performed by experienced laboratory personnel using controlled protocols, false-negative (caused primarily by endogenous polymerase inhibitors) and false-positive results (due to contamination) can generally be avoided, achieving sensitivity and specificity close to 100%. In the clinical practice, the use of molecular testing for the diagnosis of tuberculosis, in combination with "classic" diagnostic tools, can greatly enhance the diagnostic ability of pulmonary clinicians, particularly in paucibacillary infections and in patients with atypical presentation, such as immunodeficient individuals.