The greenhouse gas nitrous oxide (N2O) can be both formed and consumed by microbial processes in the soil. As these processes fractionate strongly in favour of 14N and 16O, 15N and 18O gradients of N2O in the soil profile may elucidate patterns of N2O formation, consumption or emission to the atmosphere. We present the first in situ data of such gradients over time for a mesic typic Haplaquod seeded with potatoes (Solanum tuberosum L.). On two adjacent fields in 2002 and 2003, topsoil N2O fluxes were measured and the soil atmosphere was regularly sampled for N2O concentrations, 15N and 18O signatures of N2O at depths of 18, 48 and 90 cm during 400 days. During the entire sampling period, the N2O concentrations were the highest and the 15N signatures the lowest in the subsoil (48 or 90 cm depth) as compared with the topsoil, indicating production of N2O in the subsoil. For 15N, differences greater than 30 between topsoil and subsoil on the same date were regularly observed. The highest N2O concentration of 100385 L m-3 at 90 cm depth on 1 July 2003, was preceded by the lowest 15N value of -43.5 one week earlier. This was followed by a 150-day general decrease of N2O concentrations at 90 cm depth to 1723 L m-3 and a simultaneous enrichment of 15N to +7.1, mostly without a significant topsoil flux. There was a negative logarithmic relationship between N2O concentration at 90 cm depth and its 15N signature. This relationship indicated a 15N signature of -40 to -45 during the production of N2O in the subsoil, and a subsequent enrichment during the consumption of N2O. We conclude that the isotopic signature of the N2O topsoil flux is the result of various processes of consumption and production at different depths in the soil profile. It is therefore not a reliable estimator for the overall 15N signature of N2O in the soil atmosphere, nor for indirect losses of N2O to the environment. Therefore, these findings will pose a further challenge to ongoing efforts to draw up a global isotopic budget for N2O.