The INNO-LiPA Mycobacteria kit has been developed for detecting mycobacteria in liquid and solid cultures through amplification of the 16S-23S rRNA mycobacterial spacer region and the use of species-specific probes. The aim of this study was to verify the possible direct use of the kit on clinical samples. The study was performed retrospectively on a total of 129 specimens (104 pulmonary and 25 extrapulmonary) and the results were compared to those obtained from culture. For pulmonary specimens, the overall clinical sensitivity of INNO-LiPA Mycobacteria kit was 79.5% and its specificity 84.6%. For extrapulmonary samples, the kit had an overall clinical sensitivity of 71.4%. In both cases no false positive results were found.