Pseudomonads are wide-spread bacteria known for their degradation abilities. Bacteria of the genus are ubiquitous in soil, water and many other natural habitats. Their main importance lies in modification of enviroment (geochemical cycles, biodegradation). Some members of the genus are important pathogens of plants, animals and human. The genus Pseudomonas sensu stricto comprises 160 validly described species and 8 subspecies, at present. Classification of environmental Pseudomonas isolates is often complicated due to heterogeneity and genetic variability of the species. Analysis of intact bacteria cells by Matrix-Assisted Laser Desorption Ionisation Time-of-Flight Mass Spectrometry (MALDI-TOF MS) has been shown to provide characteristic peptide/protein profiles which enable rapid identification of the microorganisms.1 In our work, sample preparation procedures were optimised for this group of Gram-negative bacteria including culture preparation and conditions of MALDI-MS analysis. MS data were processed with software developed at the Department of Informatics. The processing step involves three separate procedures: i) peak recognition and quantification, ii) calculation of inter-spectra similarity values based on selected metrics (shared peak count and its modifications, vector cosine and euclidian distance) and iii) clustering and classification based on the obtained similarity data. Results show our ability to distinguish subtle differences between the samples, well below the species level. MALDI MS has proven as a useful technique for fast taxonomic characterisation of pseudomonads. 1. C. Fenselau, P.A. Demirev, Mass Spectrom. Rev. 20, 157 (2001).