Isotopologue values were used to quantify the contribution of denitrification to nitrous oxide (N2O) flux in agricultural and early successional fields in southwest Michigan. Nitrous oxide-δ15N and δ18O values were poor estimators of microbial origins compared to site preference (SP) (difference in δ15N between the outer and central N atoms of N2O). Site preference was used to evaluate the importance of denitrification (including nitrifier denitrification) in N2O production. Average flux-weighted SP values for each field ranged between 2.9 and 14.6‰ and, on the basis of SP values for N2O production from denitrification (0‰) and collectively for nitrification and fungal denitrification (37‰), these values indicate that between 61 and 92% of N2O originated from denitrification. Reduction of N2O ranged from undetectable to as much as 50% of production; and because reduction increases SP, our results underestimate the percentage of N2O from bacterial denitrification. The SP values in our study clearly indicate that denitrification is the predominant source of soil-derived N2O.