Strong accumulations of N2O at oxyclines are some of the most conspicuous features of the world's oceans. However, the origin of these maxima, and the relative contribution of nitrification and denitrification in N2O cycling, remains unclear. In order to gain insight into the importance of denitrification and factors regulating N2O cycling at upper oxyclines in the eastern South Pacific, the production and consumption of N2O by denitrification were measured using a classical acetylene method under induced anoxia with the addition of an electron acceptor (nitrite) and donors (sodium acetate and glucose). The results indicated that decreased O2 clearly affected the ratio in which N2O is reduced to N2 at the midoxycline (∼40 m depth) and at the oxycline's base (∼80 m depth). Under induced anoxia, higher N2O production (from NO2 to N2O of 67.2 nM d–1) occurred at 40 m depth, with half of the total quantity being consumed by denitrification (from N2O to N2 of 32 nM d–1); conversely, 100% of the N2O was reduced to N2 at 80 m depth. In comparison with previously reported results at the base of the oxycline at an offshore station, the addition of NO2 (as sodium nitrite) along with dissolved organic carbon (as sodium acetate and glucose) doubled the net N2O production by denitrification (∼20 nM d–1). Our results suggest that decreasing O2 levels along with an increased availability of NO2 and organic compounds in the upper oxycline may impact the N2O/N2 ratio and, therefore, the N2O efflux to the atmosphere.