Most nitrogen (N) assimilation in lake and marine ecosystems is often subsequently released via autochthonous dissolved organic nitrogen (DON) production, but autochthonous DON production has yet to be quantified in flowing waters. We measured in-stream DON production following 24 h 15N-nitrate (NO-3) tracer additions in 36 headwater streams, a subset of sites from the second Lotic Intersite Nitrogen eXperiment. Streams were located in five North American ecoregions and drained basins dominated by native vegetation, agriculture, or urban land use. Using a two-compartment model, we could quantify DON production in 15 streams as a function of DO15N derived from 15N tracer in biomass compartments. The streams with detectable DON production had higher % modified land use (agriculture + urban) in their basins than did streams with undetectable DON production. Median DON production represented 8% of total NO-3 uptake when we used N biomass estimates based on N assimilated over 1 d (measured directly from the 15N additions). Median DON production was 17% of total NO-3 uptake when we used N assimilated over 42 d (extrapolated from previous 15N tracer studies). Variation in DON production was positively correlated with ecosystem respiration, indicating that stream heterotrophy may influence DON production. In-stream DON production was similar in magnitude to stream denitrification and nitrification, indicating that the production of autochthonous DON can represent a substantial transformation of stream N. Our results confirm that headwater streams can quickly convert inorganic N into organic forms, although the ultimate fate of DON remains unclear.