The bacteriological methods traditionally used in the diagnosis of acute respiratory infections (ARI) have limited sensitivity (culture, direct antigen detection, etc.) or require long periods to obtain results (appearance of antibodies). In the last few years, nucleic acid amplification techniques (NAAT) have been developed that allow pathogen-specific genetic targets to be detected in clinical samples. These techniques have been proven to be more sensitive than culture or direct detection and, unlike serological tests, are effective in the acute phase of the infection. However, NAAT also have certain limitations, such as the occasional presence of amplification inhibitors in clinical samples, the persistence of Mycoplasma pneumoniae or Chlamydophila pneumoniae in the mucosa of some individuals, and the lack of discrimination between pathogen infection and colonization in bacteria forming part of normal respiratory tract flora (Streptococcus pneumoniae). Recently developed real-time NAAT have raised expectations that some of these obstacles will be resolved, since these techniques allow bacterial load to be quantified. In the etiological diagnosis of ARI due to S. pneumoniae, the use of NAAT is still in an experimental phase. In M. pneumoniae and C. pneumoniae, combining NAAT with serological tests could potentially improve diagnosis. NAAT show good sensitivity and specificity in the detection of Legionella; however, the practical utility of these techniques should be weighed against that of antigenuria. NAAT provide advantages over other techniques in Bordetella pertussis. At present, these techniques are not useful in the diagnosis of Coxiella burnetii acute infections.