Nitrous oxide (N O), 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 N O emissions, often including AOB as the main N O 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 N O 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 N O production from mixed cultures is essential. The proposed N O 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 N O production. Importantly, the individual contribution of autotrophic and heterotrophic to the total N O pool could not be unambiguously elucidated solely based on bulk N O measurements. Data on NO would increase the practical identifiability of N O production pathways. Biotechnol. Bioeng. 2017;114: 132-140. © 2016 Wiley Periodicals, Inc.