Six species have been described, based on host preferences, metabolism, culture and antigenic features, including the two most recent species, isolated from seals and cetaceans, but most human cases remain to be caused by B. melitensis and B. abortus. However, DNA-DNA hybridization shows a high homology between strains, indicating that current species should be rather considered as subspecies corresponding to evolutionary lineages adapted to specific hosts. Classically, biphasic blood cultures such as the Ruiz-Castan˜eda method were used to isolate brucellae from blood and bone marrow. Now, most laboratories use continuous-monitoring automated blood culture systems, which can shorten the time to isolation and have been shown to be highly sensitive. Nevertheless, subculture is necessary to identify the microorganism, and brucellae may require 2–3 days to grow on chocolate or blood agar. Rapid automated bacterial identification systems must be interpreted with caution, because brucellae have been misidentified with some of these systems. PCR have shown high sensitivity and specificity, but its use CP-690550 side effects remains infrequent, mainly due to standardization problems. MALDI-TOF mass spectrometry has been suggested as a fast and reliable method for bacterial identification, based on protein profiles characteristic of each microorganism. Databases have been developed that include the main pathogenic microorganisms, thus allowing the use of this method in routine bacterial identification from plate culture. This is an important problem for the routine use of MALDI-TOF MS for the direct diagnosis of blood cultures in countries where brucellosis is still frequent. The aim of our study was to identify and differentiate Brucella species by MALDI-TOF MS, combining MALDI-TOF MS with dedicated bioinformatics and statistical methods. Initial spectra from three type strains of B. melitensis, five type strains of B. abortus and one type strain of B. suis, B. canis, B. ceti and B. pinnipedialis were used to set up database entries for re-identification of Brucella strains. This database was evaluated with 131 blind-coded Brucella clinical isolates previously identified by conventional methods. We also tested the reliability of this method for identifying brucellae directly from blood cultures. Brucellosis remains a serious problem in wide areas around the world. Symptoms are nonspecific. This lack of specificity of symptoms may delay the diagnosis for weeks. Though mortality rate is currently low, it remains a severe disease, and complications such as epididymoorchitis, arthritis, and CNS complications are not infrequent.