Nitrogen (N) fixation is fueling planktonic production in a multitude of aquatic environments. In meso- and poly-haline estuaries, however, the contribution of N by pelagic N₂ fixation is believed to be insignificant due to the high input of N from land and the presumed absence of active N₂-fixing organisms. Here we report N₂ fixation rates, nifH gene composition and nifH gene transcript abundance for key diazotrophic groups over 1 year in two contrasting, temperate, estuarine systems: Roskilde Fjord (RF) and the Great Belt (GB) strait. Annual pelagic N₂ fixation rates averaged 17 and 61 mmol N m(-2) per year at the two sites, respectively. In RF, N₂ fixation was mainly accompanied by transcripts related to heterotrophic (for example, Pseudomonas sp.) and photoheterotrophic bacteria (for example, unicellular diazotrophic cyanobacteria group A). In the GB, the first of two N₂ fixation peaks coincided with a similar nifH-expressing community as in RF, whereas the second peak was synchronous with increased nifH expression by an array of diazotrophs, including heterotrophic organisms as well as the heterocystous cyanobacterium Anabaena. Thus, we show for the first time that significant planktonic N₂ fixation takes place in mesohaline, temperate estuaries and that the importance of heterotrophic, photoheterotrophic and photosynthetic diazotrophs is clearly variable in space and time.