Determination of N₂O-reductase activity in soil is important for understanding the microbial regulation of nitrous oxide (N₂O) concentrations in soil. Unfortunately, there are no easily applicable and accurate methods for determining N₂O-reductase activity, which frustrates the understanding of the mechanisms that control soil management effects on denitrification and N₂O emissions. The objectives of the study reported here were (i) to define the optimal experimental conditions for the determination of potential N₂O-reductase activity in soil, (ii) to compare the direct-N₂ method with the acetylene inhibition technique, and (iii) to investigate the effects of long-term nitrogen (N) fertilization on the potential N₂O-reductase activity. 

　　Various substrate concentrations, water/soil ratios, incubation times, temperatures and pH values were tested to find the optimal conditions for the potential N₂O-reductase activity in soils from two sites. Then, the potential N₂O-reductase activity was determined under optimal conditions (10 g soil, 10 ml buffer solution, pH 9, 40 °C, 100 ppmv N₂O) in soils from the long-term N fertilization experiment. There were significant differences between soils in potential N₂O-reductase activity, but the optimal experimental conditions were similar. The acetylene inhibition technique underestimated N₂O-reductase activity in soil relative to the direct-N₂ method, especially in the treatment where fertilizer N was withheld for 15 yrs. We recommend that the optimal experimental conditions for the determination of the potential N₂O-reductase activity are established also for other soils. More studies are needed to fully understand the interactive effects of long-term N fertilization on nosZ gene expression and N₂O-reductase activity in soils.