Integration of perennial filter strips (PFS) into the toeslopes of agricultural watersheds may decrease downstream nitrate (NO₃) losses. However, long-term NO₃ removal depends on the relative importance of several NO₃ sinks in the PFS. Plant biomass and labile soil organic matter (SOM) are temporary NO₃ sinks, while stable SOM is a long-term, but potentially finite, NO₃ sink. In contrast, denitrification is a permanent NO₃ sink. We investigated the relative importance of these NO₃ sinks in PFS at the toeslope of row crop watersheds in Iowa. Using 25- × 30-cm in situ mesocosms, we added ¹⁵NO₃ to PFS soils and quantified ¹⁵NO₃–N recovery in plant biomass and SOM after one growing season. Further, we compared ¹⁵NO₃–N recovery in particulate (relatively labile) and mineral-associated (relatively stable) SOM in mesocosms with and without growing perennial vegetation. To determine the potential importance of denitrification, we compared denitrification enzyme activity in soils from paired watersheds with and without PFS. Transfer of ¹⁵NO₃–N into labile and stable SOM pools was rapid and initially independent of growing vegetation. However, SOM and plant biomass were both relatively minor NO₃ sinks, accounting for 15NO₃–N inputs. Denitrification enzyme activity data indicated that dissolved organic carbon derived from perennial vegetation increased potential denitrifier activity in PFS soils compared with row crop soils. Together, these results constrain SOM and plant biomass as NO₃ sinks and indicate that denitrification was the most important NO₃sink in perennial filter strips over one growing season.