Nitrogen fixation using the acetylene reduction assay was investigated at a variety of sediment sites in the River Eden estuary, Scotland. There was a gradient of activity, with lowest rates on mud-flats at the mouth (< 0'1 nmol C2H4 g dry wt sediment -1 h -1) to highest rates on saltmarshes at the head of the estuary (up to 13 nmols C2H4 g dry wt sediment -1 h-l). The activity found on the saltmarshes was light dependent and confined to the top 2 cm sediment whilst on the mud-flats the observed activity took place in the dark and was distributed throughout the top 4 cm. Seasonally, highest rates were found during the summer months on both marshes and mud-flats but high rates were maintained on the marshes during winter. Acetylene reduction was observed to take place over the range of sediment temperatures, water content and salinities normally found in the estuary. Dark acetylene reduction was found associated with the roots of Zostera, Salicornia and Juneus and also Zostera leaves. High rates were also found associated with the thalli of Enteromorpha and Ulva. Over the 12-month study period the total calculated quantity of nitrogen fixed throughout the estuary (3'47 × 103 kg N) was of the same order as the amount of nitrogen bound in the Enteromorpha biomass and the dissolved biorganic nitrogen in the top 3 cm sediment. The quantity of nitrogen fixed is low relative to the gross freshwater input but the continuous supply to the sediments and the localized high rates associated with the macro-primary producers suggest a possible role for nitrogen fixation in the nitrogen budget of this estuary.