Nitrous oxide (N2O) is an important and strong greenhouse gas in the atmosphere and part of a feed-back loop with climate. N2O is produced by microbes during nitrification and denitrification in terrestrial and aquatic ecosystems. The main sinks for N2O are turnover by denitrification and photolysis and photo-oxidation in the stratosphere. The position of the isotope 15N in the linear N = N = O molecule can be distinguished between the central or terminal position (isotopomers of N2O). It has been demonstrated that nitrifying and denitrifying microbes have a different relative preference for the terminal and central position. Therefore, measurements of the site preference in N2O can be used to determine the source of N2O i.e. nitrification or denitrification. Recent instrument development allows for continuous (on the order of days) position dependent δ15N measurements at N2O concentrations relevant for studies of atmospheric chemistry. We present results from continuous incubation experiments with denitrifying bacteria, Pseudomonas fluorescens (producing and reducing N2O) and P. chlororaphis (only producing N2O). The continuous position dependent measurements reveal the transient pattern (KNO3 to N2O and N2, respectively), which can be compared to previous reported site preference (SP) values. We find bulk isotope effects of −5.5 ‰ ± 0.9 for P. chlororaphis. For P. fluorescens, the bulk isotope effect during production of N2O is −50.4 ‰ ± 9.3 and 8.5 ‰ ± 3.7 during N2O reduction. The values for P. fluorescens are in line with earlier findings, whereas the values for P. chlororaphis are larger than previously published δ15Nbulk measurements from production. The calculations of the SP isotope effect from the measurements of P. chlororaphis result in values of −6.6 ‰ ± 1.8. For P. fluorescens, the calculations results in SP values of −5.7 ‰ ± 5.6 during production of N2O and 2.3 ‰ ± 3.2 during reduction of N2O. In summary, we implemented continuous measurements of N2O isotopomers during incubation of denitrifying bacteria and believe that similar experiments will lead to a better understanding of denitrifying bacteria and N2O turnover in soils and sediments and ultimately hands-on knowledge on the biotic mechanisms behind greenhouse gas exchange of the Globe.