Nitrous oxide (N2O) is a major greenhouse gas that is mainly produced but also reduced by microorganisms in soils. We determined factors for N and O isotope fractionation during the reduction of N2O to N2 in soil in a flow-through incubation experiment. The absolute value of the fractionation factors decreased with increasing reaction rate constant. Reaction rates constants ranged from 1.7 10−4 s−1 to 4.5 10−3 s−1. The minimum, maximum and median of the observed fractionation factors were for N −36.0‰, −1.0‰ and −9.3‰ and for O −74.0‰, −6.9‰ and −26.3‰, respectively. The ratio of O isotope fractionation to N isotope fractionation was 2.4 ± 0.3 and it was independent from the reaction rate constants. This leads us to conclude that fractionation factors are variables while their ratio in this particular reaction might be a constant. Copyright © 2007 John Wiley & Sons, Ltd.