A nested polymerase chain reaction (PCR) procedure was devised for identification of mycobacteria. The outer reaction exploiting genus-specific sequences on the 16S rRNA gene was able to amplify specifically strains of the genus Mycobacterium. The identification of Mycobacterium tuberculosis complex, Mycobacterium avium and Mycobacterium intracellulare was accomplished by selective reamplification of the outer PCR product in three distinct inner amplifications exploiting species-specific primers mapping to a hypervariable region of mycobacterial 16S rRNA. Detection of mycobacteria, other than those for which species-specific primers were used, was accomplished by adding a supplementary genus-specific upper primer to one of the inner reactions. Specificity of amplification was confirmed for clinical isolates and reference strains of different mycobacterial species with the exception of a M. intracellulare type 7 strain which was recognized as M. avium. The amplification protocol presented thus provides a reliable and cost-effective way for identification of clinically relevant mycobacteria.