BACKGROUND: Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) has been used for the identification of bacteria worldwide. To our knowledge, the evaluation of MALDI-TOF MS for the identification of bacteria in Korea has not been studied. In this paper we compared the identification results of aerobic bacteria using MALDI-TOF MS to those results using conventional biochemical methods. METHODS: We evaluated the performance of a MALDI-TOF MS system (Bruker Daltonics, Leipzig, Germany) on consecutive aerobic isolates collected from January to February of 2011 which were identified using conventional methods (biochemical testing and commercial identification kits). Either directly smearing onto the target plate or protein extraction methods were additionally used if no reliable or discordant results were obtained. RESULTS: Among 523 isolates tested, 506 (97%) isolates had valid scores (> or =2.0), 11 (2%) isolates gave intermediate scores (1.7< or = score <2.0), and 6 (1%) isolates yielded no reliable identification (score <1.7). Of the 506 valid results (score > or =2.0) by MALDI-TOF MS, the identification matched at the species level in 486 (96%) isloates, matched at the genus level in 17 (3%) isloates, and was discordant at the genus and species levels in 3 (1%) isloates. CONCLUSION: The overall matching rate at the species level of MALDI-TOF MS was very high. When MALDI-TOF MS did not yield reliable results by direct smear, additional direct smears or protein extraction methods could be used to obtain better results. Our results showed that MALDI-TOF MS is a very useful method for the identification of aerobic bacteria isolated in clinical microbiology laboratories.