Within the past years MALDI-TOF mass spectrometry has become a powerful tool for the species identification of cultured bacteria and fungi. In the present study, the implementation of MALDI-TOF mass spectrometry in a commercial high throughput laboratory is described. The impact of different growth conditions on the identification results was evaluated. Although slight differences in MALDI-TOF spectra of E. coli and S. aureus strains cultured on blood agar for various periods (5, 18, 24 and 48 hours) were noticed, reliable species identification was obtained for all periods. The same was true when E. coli and S. aureus strains were cultured for 18 hours on various solid media. Reliable identification was also achieved when fungi were cultured on solid and in liquid media (Sabouraud bouillon). Moreover, growth of fungi in bouillon resulted in accelerated identification. MALDI-TOF mass spectrometry also allowed reliable identification of microorganisms from positive blood culture samples. In total, 2,900 specimens (234 different species) predominantly derived from clinical samples were examined. Microorganisms were cultured on solid media, in blood culture bottles and in liquid Sabouraud bouillon. 98.6% (n=2,860) of the MALDI-TOF identification results matched those of conventional methods (e.g. Gram staining, carbohydrate degradation ability, Phoenix system) and 16S rDNA PCR product sequencing. Mismatches were mainly based on missing reference spectra in the database of the analysis system. The study was performed in 2009. Due to continuous improvement of the database, even higher accuracy would be achieved when performing the study nowadays. In summary, the use of MALDI-TOF mass spectrometry in a clinical high throughput environment leads to reliable results, even when various culture conditions are used, for instance bouillons for culture of fungi. MALDI-TOF mass spectrometry is a fast and robust identification system, which is on its way to become a new standard for the idenrification of microorganisms in high throughput laboratories