The acetylene inhibition technique (AIT) is commonly used for the determination of soil denitrification potential (SDP). However, the results obtained with this technique have intrinsic uncertainties. This study aimed at quantifying these uncertainties. A silt loam topsoil from an experimental station was incubated under standard conditions for SDP measurements, with and without acetylene additions. Changes in the concentrations of N2O and N2 in the headspace of incubation vials were monitored during the incubation, using a robotized sampling and analyzing system. Our results showed that the AIT did not completely inhibit the reduction of N2O toN2. As much as 11.7% of the produced N2O was reduced into N2 in the presence of acetylene. We conclude that SDP has to be corrected when determined by the AIT. The procedures used in this study will allow the estimation of correction factors, possibly as function of soil type and environmental conditions.