Bacterial degradation pathways of fuel oxygenates such as methyl tert-butyl and tert-amyl methyl ether (MTBE and TAME, respectively) have already been studied in some detail. However, many of the involved enzymes are still unknown, and possible side reactions have not yet been considered. In Aquincola tertiaricarbonis L108, Methylibium petroleiphilum PM1, and Methylibium sp. strain R8, we have now detected volatile hydrocarbons as by-products of the degradation of the tert-alkyl ether metabolites tert-butyl and tert-amyl alcohol (TBA and TAA, respectively). The alkene isobutene was formed only during TBA catabolism, while the beta and gamma isomers of isoamylene were produced only during TAA conversion. Both tert-alkyl alcohol degradation and alkene production were strictly oxygen dependent. However, the relative contribution of the dehydration reaction to total alcohol conversion increased with decreasing oxygen concentrations. In restingcell experiments where the headspace oxygen content was adjusted to less than 2%, more than 50% of the TAA was converted to isoamylene. Isobutene formation from TBA was about 20-fold lower, reaching up to 4% alcohol turnover at low oxygen concentrations. It is likely that the putative tert-alkyl alcohol monooxygenase MdpJ, belonging to the Rieske nonheme mononuclear iron enzymes and found in all three strains tested, or an associated enzymatic step catalyzed the unusual elimination reaction. This was also supported by the detection of mdpJK genes in MTBE-degrading and isobutene-emitting enrichment cultures obtained from two treatment ponds operating at Leuna, Germany. The possible use of alkene formation as an easy-to-measure indicator of aerobic fuel oxygenate biodegradation in contaminated aquifers is discussed.The extensive use of methyl tert-butyl ether (MTBE) and related compounds as fuel oxygenates has resulted in contamination of numerous groundwater sites in the United States and Europe (13,26,33,51). Although MTBE is now banned in some countries and may be phased out in others (52), it will persist at polluted sites for a long time due to its poor biodegradability. Despite this enormous environmental prevalence, only a few strains capable of growth on MTBE, ethyl tert-butyl ether (ETBE), and tert-amyl methyl ether (TAME) have been found, and fuel oxygenate metabolism has not been elucidated in full detail (20,30,31,46). Since the pioneering works of Salanitro et al. and Steffan et al. (42,43,48), it is generally agreed that aerobic MTBE degradation proceeds via a monooxygenase-catalyzed ether cleavage resulting in formation of tert-butyl alcohol (TBA). The latter is hydroxylated to the corresponding diol, 2-methyl-1,2-propanediol (MPD), which is further oxidized to 2-hydroxyisobutyric acid (2-HIBA). This branched carboxylic acid is then introduced into common metabolic routes after isomerization to 3-hydroxybutyric acid (39). Since ETBE shares the tert-butyl structure with MTBE, it should have a similar degradation path via TBA (8, 30). The biochemistry of TAME catabol...