A proteome map of Ralstonia metallidurans strain CH34 was constructed using two-dimensional (2-D) gel electrophoresis in combination with automated Edman degradation and mass spectrometry (MS). R. metallidurans CH34 is the type-strain of a family of highly related strains characterized by their multiple resistance to millimolar amounts of heavy metals, conferred by two large plasmids. The protein content of this bacterium grown in minimal medium was separated by 2-D gel electrophoresis using various pH gradients. Protein identification was carried out via N-terminal amino acid sequencing, matrix assisted laser desorption/ionisation-time of flight-mass spectrometry (MALDI-TOF-MS) and tandem MS. So far, 224 different proteins were characterized from 352 protein spots. Although the proteome map is still not complete, one could appraise the importance of proteomics for genome analyses through (i). the identification of previously undetected open reading frames, (ii). the identification of proteins not encoded by the already sequenced genome fragments, (iii). the characterization of protein-encoding genes spanning two different contigs, enabling their merging, and (iv). the precise delineation of the N-terminus of several proteins. Finally, this map will prove a useful tool in the identification of proteins differentially expressed in the presence of different heavy metals.