Polar lipid-derived fatty acids (PLFA) commonly found in sulfate-reducing bacteria were detected in high abundance in the sediment harvested from a monitoring well of a petroleum-hydrocarbon (PHC)-contaminated aquifer. Aquifer microcosms were incubated under sulfate-reducing conditions with [methyl-14 C]toluene to determine the 14 C-mass balances and with [methyl-13 C]toluene to follow the flow of carbon from toluene into biomarker fatty acids. An aliquot was used to establish an aquifer-derived toluene-degrading sulfate-reducing consortium, which grew well in liquid medium. Whole-cell hybridization using 16S rRNA-targeted oligonucleotide probes specific for different phylogenetic levels within the sulfate-reducing bacteria was applied in order to characterize the sulfate-reducing populations in the original sediment, the aquifer microcosms, and the aquifer-derived consortium. In the aquifer microcosms, the 14 C quantification revealed that 61.6% of the [methyl-14 C]toluene was mineralized and 2.7% was assimilated. Following [methyl-13 C]toluene depletion ( 6 1 WM), the highest 13 C-enrichment was found in PLFA 16:1g5c. In addition, biomarker fatty acids characteristic for the genera Desulfobacter and Desulfobacula (cy17:0 and 10Me16:0) were also 13 C-enriched, contrary to those of other sulfate-reducing genera, e.g. Desulfovibrio and Synthrophobacter (i17:1g7c), Desulfobulbus and Desulforhabdus (15:1g6c and 17:1g6c). Although hybridization detection rates remained low, indicating low bacterial activities, 43% (aquifer sediment) and 30% (aquifer microcosm) of the total active bacteria belonged to the Desulfobacteriaceae thus supporting the PLFA-based results. Desulfobacter-species (42%), which belong to the Desulfobacteriaceae, dominated the community of the consortium. Our study showed that carbon stable isotope analysis in combination with whole-cell hybridization could link toluene degradation in aquifer microcosms to the metabolic activity of the Desulfobacter-like populations. These populations could play an important role in the clean up of aromatic PHC-contaminated aquifers. ß