Soils are an important source of N2O, which can be produced both in the nitrification and the denitrification processes. Grassland soils in particular have a high potential for mineralization and subsequent nitrification and denitrification. When ploughing long term grassland soils, the resulting high supply of mineral N may provide a high potential for N2O losses. In this work, the short-term effect of ploughing a permanent grassland soil on gaseous N production was studied at different soil depths. Fertiliser and irrigation were applied in order to observe the effect of ploughing under a range of conditions. The relative proportions of N2O produced from nitrification and denitrification and the proportion of N2 gas produced from denitrification were determined using the methyl fluoride and acetylene specific inhibitors. Irrespectively to ploughing, fertiliser application increased the rates of N2O production, N2O production from nitrification, N2O production from denitrification and total denitrification (N2O + N2). Application of fertiliser also increased the denitrification N2O/N2 ratio both in the denitrification potential and in the gaseous N productions by denitrification. Ploughing promoted soil organic N mineralization which led to an increase in the rates of N2O production, N2O production from nitrification, N2O production from denitrification and total denitrification (N2O + N2). In both the ploughed and unploughed treatments the 0–10 cm soil layer was the major contributing layer to gaseous N production by all the above processes. However, the contribution of this layer decreased by ploughing, gaseous N productions from the 10 to 30 cm layer being significantly increased with respect to the unploughed treatment. Ploughing promoted both nitrification and denitrification derived N2O production, although a higher proportion of N2O lost by denitrification was observed as WFPS increased. Recently ploughed plots showed lower denitrification derived N2O percentages than those ploughed before as a result of the lower soil water content in the former plots. Similarly, a lower mean nitrification derived N2O percentage was found in the 10–30 cm layer compared with the 0–10 cm.