Nitrous oxide (N2O), a by-product of biological nitrogen removal during wastewater treatment, is produced by ammonia-oxidizing bacteria (AOB) and heterotrophic denitrifying bacteria (HB). Mathematical models are used to predict N2O emissions, often including AOB as the main N2O producer. Several model structures have been proposed without consensus calibration procedures. Here, we present a new experimental design that was used to calibrate AOB-driven N2O dynamics of a mixed culture. Even though AOB activity was favoured with respect to HB, oxygen uptake rates indicated HB activity. Hence, rigorous experimental design for calibration of autotrophic N2O production from mixed cultures is essential. The proposed N2O production pathways were examined using five alternative process models confronted with experimental data inferred. Individually, the autotrophic and heterotrophic denitrification pathway could describe the observed data. In the best-fit model, which combined two denitrification pathways, the heterotrophic was stronger than the autotrophic contribution to N2O production. Importantly, the individual contribution of autotrophic and heterotrophic to the total N2O pool could not be unambiguously elucidated solely based on bulk N2O measurements. Data on NO would increase the practical identifiability of N2O production pathways. (C) 2016 Wiley Periodicals, Inc.