2-Ethyhexyl nitrate (2-EHN) is a major additive of fuel that is used to increase the cetane number of diesel. Because of its wide use and possible accidental release, 2-EHN is a potential pollutant of the environment. In this study, Mycobacterium austroafricanum IFP 2173 was selected from among several strains as the best 2-EHN degrader. The 2-EHN biodegradation rate was increased in biphasic cultures where the hydrocarbon was dissolved in an inert non-aqueous-phase liquid, suggesting that the transfer of the hydrophobic substrate to the cells was a growth-limiting factor. Carbon balance calculation, as well as organic-carbon measurement, indicated a release of metabolites in the culture medium. Further analysis by gas chromatography revealed that a single metabolite accumulated during growth. This metabolite had a molecular mass of 114 Da as determined by gas chromatography/mass spectrometry and was provisionally identified as 4-ethyldihydrofuran2(3H)-one by liquid chromatography-tandem mass spectrometry analysis. Identification was confirmed by analysis of the chemically synthesized lactone. Based on these results, a plausible catabolic pathway is proposed whereby 2-EHN is converted to 4-ethyldihydrofuran-2(3H)-one, which cannot be metabolized further by strain IFP 2173. This putative pathway provides an explanation for the low energetic efficiency of 2-EHN degradation and its poor biodegradability.