Using acetylene to inhibit the reduction of N 2O to N 2 is the simplest and most commonly used technique to quantify denitrification. The acetylene treatment may not always inhibit N 2O-reductase completely when soils are wet, low in NO 3 − or with a high C-to-NO 3 − ratio. Acetylene blocks nitrification as well as N 2O-reductase, so if nitrification is the source of NO 2 − or NO 3 − for denitrification, then the denitrification rate would be underestimated by the C 2H 2 inhibition technique. Acetylene can also be metabolised in soil and so enhance denitrification. We show how the 15N method for measuring denitrification can be combined with the C 2H 2 inhibition technique to check the effect of acetylene on N 2O-reductase, respiration and nitrification. No method exists, however, for the analysis of 15N in the N 2 and N 2O of headspace samples which also contain 10% v/v C 2H 2. We describe a technique to remove C 2H 2 from headspace samples with alkaline KMnO 4 prior to analysis by isotope-ratio mass spectrometry. The C 2H 2 removal procedure had no effect on the determination of N 2O concentration, or on the determination of the 15N content of N 2O and N 2. Then using data for fluxes of N 2O, N 2, (N 2O + N 2), CO 2 and the size and 15N content of the soil NO 2 − pool for a soil incubation experiment, we show how the effects of C 2H 2 can be quantitatively assessed. The data also allowed the amount of denitrification, as estimated by the C 2H 2 inhibition technique, to be compared with that measured using the 15N technique. For the soil conditions during this incubation, 10% v/v C 2H 2 completely blocked N 2O-reductase, slightly inhibited respiration and completely blocked nitrification. The average total flux of N gases as measured by 15N (4.3 nmol N g −1 h −1) was not significantly different ( P > 0.05) from the average flux measured by the C 2H 2 inhibition method (3.9 nmol N g −1 h −1). Including C 2H 2 as a treatment in the 15N method provides a direct way of confirming that some of the assumptions in the C 2H 2 inhibition technique are valid.