Equilibrium headspace analysis of toluene for δ2H isotopic composition by continuous flow compound specific isotope mass spectrometry was determined to have an accuracy and reproducibility of ±5‰. Using this analytical approach, the hydrogen isotope fractionation produced by anaerobic biodegradation of toluene was evaluated in laboratory experiments using a mixed methanogenic consortium. A large, reproducible 2H-enrichment in the residual toluene of greater than 60‰ was observed at greater than 95% degradation, reflecting the preferential biodegradation of molecules containing the light (1H) isotope. Recent studies evaluating the magnitude of carbon isotope fractionation produced during biodegradation of aromatic hydrocarbons have documented heavy isotope (13C) enrichment in the residual contaminant approximately an order of magnitude smaller than those reported here for 2H. The very large isotopic enrichment in 2H suggests that under anaerobic conditions compound specific hydrogen isotope analysis may provide a more reliable means of validating intrinsic bioremediation of aromatic hydrocarbons than stable carbon isotope analysis. Combined application of stable carbon and hydrogen isotope analysis in an anaerobic groundwater has the potential to provide two important diagnostic tools. Relatively insensitive to biodegradation by mixed consortia, stable carbon isotope values may provide information about different sources of contaminant, while hydrogen isotope values provide an assessment of the degree of attenuation due to biodegradation.