Abstract The identification of metabolically active microbial key players is fundamental for understanding the structure and functions of contaminant‐degrading communities. The metabolic activity can be analysed by feeding the microbial culture with stable‐isotope‐labelled substrates and subsequently tracing their incorporation into the biomass. In this paper we present a method which is able to detect the incorporation of stable isotopes from the substrate into the proteins of a benzene‐metabolising microorganism. Pseudomonas putida strain ML2 was grown under aerobic conditions with the substrates 12 C‐benzene, 13 C‐benzene or 15 N‐ammonium and 12 C‐benzene. Proteins of these cultures were resolved by two‐dimensional gel electrophoresis (2‐DE) and corresponding protein spots were subjected to matrix‐assisted laser ionization/desorption mass spectrometric (MALDI‐MS) analysis. The proteins of the 12 C‐sample were identified by peptide mass fingerprinting (PMF) as well as by tandem mass spectrometric (MS/MS) measurements. The 13 C‐ or 15 N‐content of the peptides from the labelling experiments was determined by MALDI‐MS/MS. The incorporation of heavy isotopes into the proteins from cultures grown on 13 C‐benzene and 15 N‐ammonium was determined based on the mass differences between labelled and non‐labelled peptides as well as on the isotopic distribution of the y 1 ‐ion of arginine. The method we present here principally allows the unravelling of the carbon and nitrogen flow not only in pure cultures, but also in microbial communities consisting of many microbial species. Copyright © 2008 John Wiley & Sons, Ltd.