The rapid development and availability of a variety of new molecular genetic technologies present the clinician with an array of options for the accurate diagnosis of infectious diseases. This is particularly the case for tuberculosis, since molecular methods have been rapidly introduced into all working areas of the mycobacteriology laboratory. Nucleic acid amplification methods to detect Mycobacterium tuberculosis in clinical specimens are increasingly used as a tool to diagnose tuberculosis. The bulk of recently available data from clinical evaluations performed under routine laboratory conditions indicate that these molecular methods are rapid and sensitive, but yet inferior, to culture with regard to sensitivity and specificity. Therefore, until gene amplification tests have proved to be reliable and quality control procedures exist, their clinical validity remains controversial. Consequently, definition of selected clinical applications of gene amplification to routine diagnosis of tuberculosis is important and need to be discussed. This review will focus on the clinical role of molecular methods in the direct detection and diagnosis of M. tuberculosis in clinical samples. In addition, molecular genetic approaches designed to determine drug susceptibility and to discriminate strains below the species level will be outlined and discussed in terms of their current and future clinical applicability.