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The long-term efficacy of stormwater treatment systems requires continuous pollutant removal without substantial re-release. Hence, the division of incoming pollutants between temporary and permanent removal pathways is fundamental. This is pertinent to nitrogen, a critical water body pollutant, which on a broad level may be assimilated by plants or microbes and temporarily stored, or transformed by bacteria to gaseous forms and permanently lost via denitrification. Biofiltration systems have demonstrated effective removal of nitrogen from urban stormwater runoff, but to date studies have been limited to a 'black-box' approach. The lack of understanding on internal nitrogen processes constrains future design and threatens the reliability of long-term system performance. While nitrogen processes have been thoroughly studied in other environments, including wastewater treatment wetlands, biofiltration systems differ fundamentally in design and the composition and hydrology of stormwater inflows, with intermittent inundation and prolonged dry periods. Two mesocosm experiments were conducted to investigate biofilter nitrogen processes using the stable isotope tracer 15NO3(-) (nitrate) over the course of one inflow event. The immediate partitioning of 15NO3(-) between biotic assimilation and denitrification were investigated for a range of different inflow concentrations and plant species. Assimilation was the primary fate for NO3(-) under typical stormwater concentrations (∼1-2 mg N/L), contributing an average 89-99% of 15NO3(-) processing in biofilter columns containing the most effective plant species, while only 0-3% was denitrified and 0-8% remained in the pore water. Denitrification played a greater role for columns containing less effective species, processing up to 8% of 15NO3(-), and increased further with nitrate loading. This study uniquely applied isotope tracing to biofiltration systems and revealed the dominance of assimilation in stormwater biofilters. The findings raise important questions about nitrogen release upon plant senescence, seasonally and in the long term, which have implications on the management and design of biofiltration systems.