In order to clarify the incorporating mechanisms of NO3– under high N deposition environment, we monitored NO3– isotope values in deposition processes along the canopy–soil continuum in a suburban forest of Tokyo. We used stable isotopes of N and oxygen to trace the incorporation of NO3– in two types of tree stands: a Cryptomeria japonica plantation (CJ) and a natural secondary forest of Quercus acutissima (QA). Gross mineralization and nitrification rates were also measured by the pool dilution method using 15N tracers. The range in the δ18O values of rain and throughfall NO3– was large (65–70‰) but decreased rapidly to 2–5‰ in soil waters. At the QA site, δ18O values of stem flow NO3– decreased to 40‰ during several rain events, especially in the growing season. Atmospheric NO3– deposition was effectively replaced by microbially produced NO3– mainly in the organic horizon and surface part of the mineral soils. Gross rate observation showed that microbial activity, including both immobilization and nitrification in organic horizons, contributed to incorporate atmospheric NO3– quickly into the internal microbial N cycle. Additionally, we found an evidence of microbial nitrification in the tree surface of the QA stand in summer.