Nitrous oxide (N<sub>2</sub>O) is an important and strong greenhouse gas in the atmosphere and part of a feed-back loop with climate. N<sub>2</sub>O is produced by microbes during nitrification and denitrification in terrestrial and aquatic ecosystems. The main sinks for N<sub>2</sub>O are turnover by denitrification and photolysis and photo-oxidation in the stratosphere. The position of the isotope <sup>15</sup>N in the linear N = N = O molecule can be distinguished between the central or terminal position (isotopomers of N<sub>2</sub>O). 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 N<sub>2</sub>O can be used to determine the source of N<sub>2</sub>O i.e. nitrification or denitrification. Recent instrument development allows for continuous (on the order of days) position dependent <i>δ</i><sup>15</sup>N measurements at N<sub>2</sub>O concentrations relevant for studies of atmospheric chemistry. We present results from continuous incubation experiments with denitrifying bacteria, <i>Pseudomonas fluorescens</i> (producing and reducing N<sub>2</sub>O) and <i>P. chlororaphis</i> (only producing N<sub>2</sub>O). The continuous position dependent measurements reveal the transient pattern (KNO<sub>3</sub> to N<sub>2</sub>O and N<sub>2</sub>, respectively), which can be compared to previous reported site preference (SP) values. We find bulk isotope effects of −5.5 ‰ ± 0.9 for <i>P. chlororaphis</i>. For <i>P. fluorescens</i>, the bulk isotope effect during production of N<sub>2</sub>O is −50.4 ‰ ± 9.3 and 8.5 ‰ ± 3.7 during N<sub>2</sub>O reduction. The values for <i>P. fluorescens</i> are in line with earlier findings, whereas the values for <i>P. chlororaphis</i> are larger than previously published <i>δ</i><sup>15</sup>N<sub><i>bulk</i></sub> measurements from production. The calculations of the SP isotope effect from the measurements of <i>P. chlororaphis</i> result in values of −6.6 ‰ ± 1.8. For <i>P. fluorescens</i>, the calculations results in SP values of −5.7 ‰ ± 5.6 during production of N<sub>2</sub>O and 2.3 ‰ ± 3.2 during reduction of N<sub>2</sub>O. In summary, we implemented continuous measurements of N<sub>2</sub>O isotopomers during incubation of denitrifying bacteria and believe that similar experiments will lead to a better understanding of denitrifying bacteria and N<sub>2</sub>O turnover in soils and sediments and ultimately hands-on knowledge on the biotic mechanisms behind greenhouse gas exchange of the Globe.