The ethyl tert-butyl ether (ETBE) degradation capacity and phylogenetic composition of five aerobic enrichment cultures with ETBE as the sole carbon and energy source were studied. In all cases, ETBE was entirely degraded to biomass and CO2. Clone libraries of the 16S rRNA gene were prepared from each enrichment. The analyses of the DNA sequences obtained showed different taxonomic compositions with a majority of Proteobacteria in three cases. The two other enrichments have different microbiota with an abundance of Acidobacteria in one case, whereas the microbiota in the second was more diverse (majority of Actinobacteria, Chlorobi and Gemmatimonadetes). Actinobacteria were detected in all five enrichments. Several bacterial strains were isolated from the enrichments and five were capable of degrading ETBE and/or tert-butyl alcohol (TBA), a degradation intermediate. The five included three Rhodococcus sp. (IFP 2040, IFP 2041, IFP 2043), one Betaproteobacteria (IFP 2047) belonging to the Rubrivivax/Leptothrix/Ideonella branch, and one Pseudonocardia sp. (IFP 2050). Quantification of these five strains and two other strains, Rhodococcus sp. IFP 2042 and Bradyrhizobium sp. IFP2049, which had been previously isolated from one of the enrichments was carried out on the different enrichments based on quantitative PCR with specific 16S rRNA gene primers and the results were consistent with the hypothesized role of Actinobacteria and Betaproteobacteria in the degradation of ETBE and the possible role of Bradyrhizobium strains in the degradation of TBA.