ABSTRACT: A perfusion technique for measuring nitrogen fixation in the eelgrass rhizosphere was developed and used to investigate the patterns and controls of nitrogen fixation in sediment cores containing live eelgrass Zostera marina plantscollected from the Limfjord, Denmark. Detailed temporal and spatial patterns of acetylene reduction were consistent with the hypothesis that heterotrophic nitrogen fixation in the eelgrass rhizosphere is stimulated by organic root exudates derived fromplant photosynthesis. Nitrogen fixation activity was approximately 3x higher in vegetated than unvegetated sediments, and showed strong seasonal patterns and differences in light-dark incubations that corresponded to variations in plant productivity.Rates in both the light and dark were lowest in the winter months, and increased steadily through the spring and summer to a peak activity in August that coincided with the maximum eelgrass aboveground biomass. Light-incubated cores had significantlyhigher rates (25 to 40%) than those incubated in the dark during the growth season, and this difference was greatest during the summer months when plant productivity was highest. Nitrogen fixation activity also showed strong (5-fold) spatial variationwith depth in the eelgrass root zone, with highest rates corresponding to the largest root+rhizome biomass and correlating with seasonal changes in belowground biomass distribution. Additions of glucose or NH₄⁺ showed that thenitrogen-fixing bacteria were limited by organic substrate and were not sensitive to NH₄⁺ concentrations. Molybdate additions indicated that sulfate reducers were responsible for about 25% of the nitrogen fixation activity in theeelgrass rhizosphere. Overall, daily nitrogen fixation rates integrated to a depth of 14 cm in the sediment (1 to 6 mg N m^-2 d^-1) were comparable to rates measured in other temperate seagrass meadows, but were lower than thosedetermined for tropical seagrass beds.