A toluene-degrading microbial consortium was enriched directly in a BTEX-contaminated aquifer under sulfate-reducing conditions using in situ microcosms consisting of toluene-loaded activated carbon pellets. Degradation of toluene and concomitant sulfide production by the consortium was subsequently demonstrated in laboratory microcosms. The consortium was physiologically and phylogenetically characterized by isotope tracer experiments using nonlabeled toluene, [(13)C]-alpha-toluene or [(13)C(7)]-toluene as growth substrates. Cells incubated with [(13)C]-alpha-toluene or [(13)C(7)]-toluene incorporated 8-15 at.%(13)C and 51-57 at.%(13)C into total lipid fatty acids, respectively, indicating a lower specific incorporation of (13)C from [(13)C(7)]-toluene. In order to identify the toluene-assimilating bacteria, the incorporation of carbon from both [(13)C]-alpha-toluene and [(13)C(7)]-toluene into rRNA was analyzed by stable isotope probing. Time and buoyant density-resolved 16S rRNA gene-based terminal restriction fragment length polymorphism profiles, combined with cloning and sequencing, revealed that an uncultured bacterium (99% sequence similarity) related to the genus Desulfocapsa was the main toluene-degrading organism in the consortium. The ratio of the respective terminal restriction fragments changed over time, indicating trophic interactions within this consortium.