Environmental contextDissolved organic nitrogen often constitutes the largest portion of the dissolved nitrogen pool yet is a commonly overlooked nutrient source in aquatic systems. Terrestrially aged Eucalyptus camaldulensis, a common lowland leaf litter species, rapidly released proteinaceous dissolved organic nitrogen during the first 24h of leaching. The results indicate that terrestrial leaf litter may play an important role in satisfying nutrient demand within aquatic systems through both direct deposition and floodplain interactions. AbstractUnderstanding sources and forms of dissolved nitrogen is of critical importance to the management of aquatic systems worldwide. Dissolved organic nitrogen (DON) often constitutes the largest portion of the dissolved nitrogen pool, yet is commonly overlooked as a nutrient source to aquatic food webs, likely owing to its bound nature within organic material and the non-specific methods by which it is measured. In this study, we determined the protein and peptide (dissolved combined amino acid (DCAA)) contribution to DON leached from Eucalyptus camaldulensis leaves over 24h. The distribution of proteinaceous material in unleached and leached leaves was characterised using Fourier-transform infrared (FTIR) microspectroscopy to determine the likely source of DCAA within the leaf tissue. DCAAs were found to be a significant component (38.5%) of the leached DON; however, >90% of the leaf protein remained in the leaves after 24h. FTIR microspectroscopy shows that proteinaceous material is strongly partitioned to fungal colonised palisade cells in the leaf mesophyll, with evidence for depletion of this material after leaching. Comparison of leaching kinetics in the presence and absence of a microbial inhibitor (sodium azide) suggests that microbial uptake or adsorption commences within the timescales of these leaching experiments. The work shows that DON in the form of peptides and proteins leached from leaf litter is a likely source of bioavailable nutrients to in-stream and floodplain systems.