In the post-genomic era, the focus of numerous researchers has moved to studying the functional products of gene expression. In microbiology, these ‘omic’ approaches have largely been limited to pure cultures of microorganisms. Consequently, they do not provide information on gene expression in a complex mixture of microorganisms as found in the environment. Our method enabled the successful extraction and purification of the entire proteome from a laboratory-scale activated sludge system optimized for enhanced biological phosphorus removal, its separation by two-dimensional polyacrylamide gel electrophoresis and the mapping of this metaproteome. Highly expressed protein spots were excised and identified using quadrupole time-of-flight mass spectrometry with de novo peptide sequencing. The proteins isolated were putatively identified as an outer membrane protein (porin), an acetyl coenzyme A acetyltransferase and a protein component of an ABC-type branched-chain amino acid transport system. These proteins possibly stem from the dominant and uncultured Rhodocyclus-type polyphosphate-accumulating organism in the activated sludge. We propose the term ‘metaproteomics’ for the large-scale characterization of the entire protein complement of environmental microbiota at a given point in time.