Studies of nutrients in rivers and estuaries have generally focused on dissolved inorganic nitrogen (DIN), although this accounts for only a part of the total nitrogen (TN) in these systems. Dissolved organic nitrogen (DON) has not yet been fully considered as a factor contributing to the nitrogen pool in aquatic systems. Advances in the analytical determination and characterisation of DON have identified that it is potentially biologically available to phytoplankton and bacteria. Very few studies of DON have been conducted on UK rivers and estuaries therefore little is known of the concentrations, proportion of TN, seasonal variations and DON load entering estuarine waters from riverine sources. An 18 month survey of the lower reaches of the River Test and upper estuary were conducted between July 2001 and December 2002 to investigate the temporal variation in nutrient concentrations. DON concentrations up to 152 μM were measured in saline samples and concentrations of up to 100 μM were recorded in freshwater samples from the River Test. DON was the second largest TN component, contributing up to 7 % of TN in the river and 13 % of TN in the upper Test estuarine waters. DON showed no apparent relationship with season, flow or salinity, whereas dissolved organic carbon (DOC) concentration showed some seasonal changes. Concentrations of DON were reduced by tidal salt marshes and sewage treatment works were a source of DOC to the river. The estimated DON load entering the Test estuary in 2002 was 1.9 x 104 mol km-2 yr-1, one order of magnitude lower than the nitrate load. The DON area-normalised load was in good agreement with other UK river systems. The assimilation of different molecular size fractions of DON and DOC by bacteria was investigated in water collected from the lower reaches of the river. A combination of ultrafiltration and bacterial bioassays showed that the DON and DOC pools were made up of a range of molecular size classes but these did not appear to influence the growth of freshwater bacteria.