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The conventional approach to evaluate biodegradation of organic contaminants in groundwater is to demonstrate an increase in the concentration of transformation products. This approach is problematic for MTBE from gasoline spills because the primary transformation product (TBA) can also be a component of gasoline. Compound-specific stable isotope analysis may provide a useful alternative to conventional practice. Changes in the delta13C and deltaD of MTBE during biodegradation of MTBE in an anaerobic enrichment culture were compared to the delta13C and deltaD of MTBE in groundwater at nine gasoline spill-sites. The stable isotopes of hydrogen and carbon were extensively fractionated during anaerobic biodegradation of MTBE. The stable isotope enrichment factor for carbon (epsilonC) in the enrichment cultures was -13 (-14.1 to -11.9 at 95% confidence level), and the hydrogen enrichment factor (epsilonH) was -16 (-21 to -11 at 95% confidence level). The isotope enrichment factors for carbon and hydrogen during anaerobic biodegradation indicate that the first reaction is enzymatic hydrolysis of the O-Cmethyl bond. The ratio of epsilonH to epsilonC was consistent between the enrichment culture and the field site that provided the inoculum, and with the other eight sites, suggesting a common degradation pathway. Compound-specific isotope evidence is discussed in terms of its utility for monitoring in situ biodegradation, in particular, for measuring how much MTBE was degraded. For the studied field sites, significant biodegradation of the original mass of MTBE is suggested, in some cases exceeding 90%.

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