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The nitrogen dynamics of storm runoff was studied using throughfall trenches on slopes with thin soils in a white pine forest catchment near Dorset, Ontario. Hydrologic data were combined with analysis of isotopic signatures and nitrogen chemistry in throughfall, soil water, and hillslope runoff. Two hypotheses were tested: (1) macropore preferential flow pathways are a source of nitrate flushing in storm runoff, and (2) the nitrogen chemistry of subsurface storm flow is controlled by the mixing of event water fluxes via macropores with preevent soil water. Most flow occurred at the soil-bedrock interface on the slopes, and the use of 18O indicated that a considerable fraction of event water moved vertically to bedrock via preferential flow paths. Despite high levels of inorganic N in throughfall, subsurface runoff N losses during autumn storms were dominated by dissolved organic nitrogen, and little nitrate flushing occurred via preferential flow paths. Comparisons of observed NO3- and NH4+ concentrations versus concentrations predicted from the mixture of event and preevent water in subsurface flow did not support hypothesis 2 and instead indicated depletion of inorganic N. Low rates of N mineralization and negligible nitrification in surface 0–0.1 m soil during June–October suggested high biological utilization of a limited soil N supply. Laboratory experiments in which soil cores were leached with solutions containing NO3- and bromide confirmed that the organic Ae horizon was a sink for NO3-. These data suggest that the biogeochemistry of the organic horizon can regulate patterns of inorganic N loss in subsurface runoff moving by preferential flow pathways in forest soils.