The main aim of the study was to evaluate hydrogen and carbon isotope fractionation during biodegradation of benzene as a possible tool to trace the process in contaminated environments. Aerobic biodegradation of benzene by two bacterial isolates, Acinetobacter sp. and Burkholderia sp., was accompanied by significant hydrogen and carbon isotope fractionation with hydrogen isotope enrichment factors of -12.8 +/- 0.7 per thousand and -11.2 +/- 1.8 per thousand, respectively, and average carbon isotope enrichment factors of -1.46 +/- 0.06 per thousand and -3.53 +/- 0.26 per thousand, respectively. Inorganic carbon produced by Acinetobacter sp. was depleted in 13C by 3.6-6.2 per thousand as compared to the initial delta13C of benzene, while the produced biomass was enriched in 13C by 3.8 per thousand. The secondary aim was to determine isotope ratios of benzenes from different manufacturers with regard to the use of isotopes for source differentiation. While two of the four analyzed benzenes had similar delta13C values, each of them had a distinct delta2H-delta13C pair and delta2H values spread over a range of 66.5 per thousand. Thus, combined analyses of hydrogen and carbon isotopes may be a more promising approach to trace sources and/or biodegradation of benzene than measuring carbon isotopes only.