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Microbes in aquifers are present suspended in groundwater or attached to the aquifer sediment. Groundwater is often sampled at gasoline ether oxygenate (GEO)-impacted sites to assess the potential biodegradation of organic constituents. However, the distribution of GEO-degrading microorganisms between the groundwater and aquifer sediment must be understood to interpret this potential. In this study, the distribution of ethyl tert-butyl ether (ETBE)-degrading organisms and ETBE biodegradation potential was investigated in laboratory microcosm studies and mixed groundwater-aquifer sediment samples obtained from pumped monitoring wells at ETBE-impacted sites. ETBE biodegradation potential (as determined by quantification of the ethB gene) was detected predominantly in the attached microbial communities and was below detection limit in the groundwater communities. The copy number of ethB genes varied with borehole purge volume at the field sites. Members of the Comamonadaceae and Gammaproteobacteria families were identified as responders for ETBE biodegradation. However, the detection of the ethB gene is a more appropriate function-based indicator of ETBE biodegradation potential than taxonomic analysis of the microbial community. The study shows that a mixed groundwater-aquifer sediment (slurry) sample collected from monitoring wells after minimal purging can be used to assess the aquifer ETBE biodegradation potential at ETBE-release sites using this function-based concept.