ABSTRACT: Coastal microbial mats are highly diverse micro-scale ecosystems in an environment characterized by strongly fluctuating conditions. Nitrogen (N) fixation is an important process in these often N-limited ecosystems. We investigated the characteristics of N₂ fixation and the composition of the N₂-fixing (diazotrophic) community in 3 microbial mats situated along an intertidal gradient over a period of 3 yr by using the acetylene reduction assay and by analyzing more than 800 environmental nifH sequences. In 2008, nitrogenase activity (NA) in the 3 mat types differed with respect to the daily pattern and daily integrated rates (ranging from 5 to almost 150 mmol C₂H₄ m^–2 d^–1). The diazotrophic community composition in the 3 mat types was also dissimilar. The nifH clone libraries at the location that was most influenced by freshwater contained mostly cyanobacterial and alphaproteobacterial sequences, whereas the other 2 locations were dominated by gamma- and deltaproteobacterial diazotrophs. These differences were attributed to the specific environmental conditions at each location resulting from their position along the intertidal gradient and likely caused the differences in the recorded patterns of N₂ fixation. Moreover, in each of the mat types, a dramatic shift in the diazotrophic community was observed over a period of 2 to 3 yr. These shifts varied greatly between the locations. The characteristics of NA at the same location in different years also varied considerably. N₂ fixation in these microbial mats was spatially and temporally variable, which was attributed to the shifts in diazotrophic community composition along the littoral gradient and during the 3 yr of investigation.