Laboratory-scale experiments were conducted to examine the N2O emission during the denitrification process. For each of the 6 runs carried out, synthetic effluent was fed in a 10 l batch mixed liquor to investigate the effect of nitrite on N2O emission and Helium was continuously bubbled through the reactor at constant rate (0.12 l/min) to favour N2O transfer and detection. An increasing COD/NO3−-N influent ratio from 3 to 7 was firstly applied (runs 1-3). Secondly, NO2− pulse additions were performed during run 4 and 5 (10 and 20 mg N/l, respectively). Finally, the reactor was fed with influent containing both NO2− and NO3−. We showed that N2O emission was detected shortly after NO2− accumulation, few minutes after the substrate feeding. The highest emission occurred at the lower COD/NO3−-N ratio (=3) and at the higher NO2− addition (20 mg N/l). In addition, the higher nitrogen conversion to N2O gas (14.4%) was obtained with an influent containing initially both NO2− and NO3−. Our results suggest a direct effect of the NO2− concentration on the N2O emission. We have also confirmed the inhibitory effect of NO2− concentration on N2O reduction.