This study examines the relationship between dissolved inorganic nitrogen (DIN: NH4 + + NO2 − + NO3 −) and dissolved organic nitrogen (DON) in river water and groundwater in an agriculturally-dominated catchment in south-east Spain, and estimates the contribution of DON to the total dissolved nitrogen (TDN) concentrations. The studied aquifer-dependent river system consists of Quaternary alluvial sediments deposited by the Guadalquivir River and its tributaries, with both river water and groundwater in the catchment being aerobic. DON is the predominant form of nitrogen in river water (72−97% of the TDN), whereas its proportion to TDN varies considerably in groundwater (<1−99%). A seasonal pattern in the concentration of dissolved nitrate in river water was observed, whereas DON concentrations showed no significant change during the study period. The export of DON from the Guadalquivir River is approximately 2 kg N ha−1 year−1 and is an order of magnitude higher than the export of DON from pristine catchments. Dissolved nitrate concentrations in groundwater were slightly higher in winter and DON concentrations were significantly lower (p < 0.05) in summer. It is found that agricultural soils constitute the main storage of organic nitrogen in the catchment with a steady leaching of high DON concentrations into the alluvial aquifer system (mean value 19.1 mg N L−1), and so indicating that DON should not be overlooked in the nitrogen budgets of agriculturally-dominated catchments.