The value of matrix-assisted laser desorption ionization؊time of flight mass spectrometry (MALDI-TOF MS) for the identification of bacteria and yeasts is well documented in the literature. Its utility for the identification of mycobacteria and Nocardia spp. has also been reported in a limited scope. In this work, we report the specificity of MALDI-TOF MS for the identification of 162 Mycobacterium species and subspecies, 53 Nocardia species, and 13 genera (totaling 43 species) of other aerobic actinomycetes using both the MALDI-TOF MS manufacturer's supplied database(s) and a custom database generated in our laboratory. The performance of a simplified processing and extraction procedure was also evaluated, and, similar to the results in an earlier literature report, our viability studies confirmed the ability of this process to inactivate Mycobacterium tuberculosis prior to analysis. Following library construction and the specificity study, the performance of MALDI-TOF MS was directly compared with that of 16S rRNA gene sequencing for the evaluation of 297 mycobacteria isolates, 148 Nocardia species isolates, and 61 other aerobic actinomycetes isolates under routine clinical laboratory working conditions over a 6-month period. MALDI-TOF MS is a valuable tool for the identification of these groups of organisms. Limitations in the databases and in the ability of MALDI-TOF MS to rapidly identify slowly growing mycobacteria are discussed. C urrently, there are more than 170 recognized species and subspecies of mycobacteria, more than 100 Nocardia species, and several hundred other aerobic actinomycetes species distributed across approximately 16 genera (1). Some of these organisms are clinically relevant and cause a spectrum of disease presentations in humans, while others are environmental organisms that can be found as commensal organisms or in laboratory cultures as contaminants resulting from specimen collection or processing (2). Accurate identification of mycobacteria and the aerobic actinomycetes is important for patient care but can be difficult due to the low growth rates of some species, the large number of species which have small differences in genetic diversity, and the need for biosafety level (BSL) 3 facilities when unknown isolates that might be Mycobacterium tuberculosis or Mycobacterium bovis are processed. The current gold standard for the identification of mycobacteria and aerobic actinomycetes is DNA sequencing with several targets recognized as useful for the species identification of mycobacteria and aerobic actinomycetes, including the 16S rRNA gene, rpoB, secA, and hsp65 (3). However, many clinical laboratories do not have the resources to routinely perform sequencing because it is labor-intensive and technically complex.In the last few years, matrix-assisted laser desorption ionizationϪtime of flight mass spectrometry (MALDI-TOF MS) has proven to be a reliable method for the identification of a wide variety of bacteria and yeasts following growth on culture medium (4-8). Fewer studies have ...