Ethyl tert-butyl ether (ETBE) was detected at high concentration (300 mg L-1) in the groundwater below a gas-station. No significant carbon neither hydrogen isotopic fractionation of ETBE was detected along the plume. ETBE and BTEX biodegradation capacities of the indigenous microflora Pz1-ETBE and of a culture (MC-IFP) composed of Rhodococcus wratislaviensis IFP 2016, Rhodococcus aetherivorans IFP 2017 and Aquincola tertiaricarbonis IFP 2003 showed that ETBE and BTEX degradation rates were in the same range (ETBE: 0.91 and 0.83 mg L-1 h(-1) and BTEX: 0.64 and 0.82 mg L-1 h(-1), respectively) but tert-butanol (IBA) accumulated transiently at a high level using Pz1-ETBE (74 mg L-1). An on-site pilot plant (2 m(3)) filled with polluted groundwater and inoculated by MC-IFP, successfully degraded four successive additions of ETBE and gasoline. However, an insignificant ETBE isotopic fractionation was also accompanying this decrease which suggested the involvement of low fractionating-strains using EthB enzymes, but required of additional proofs. The ethB gene encoding a cytochrome P450 involved in ETBE biodegradation (present in R. aetherivorans IFP 2017) was monitored by quantitative real-time polymerase chain reaction (q-PCR) on DNA extracted from water sampled in the pilot plant which yield up to 5 x 10(6) copies of ethB gene per L-1. (C) 2011 Elsevier B.V. All rights reserved.