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Diazotrophic activity and primary production (PP) were investigated along two transects (Belgica BG2014/14 and GEOVIDE cruises) off the western Iberian Margin and the Bay of Biscay (38.8–46.5° N; 8.0–19.7° W) in May 2014 close to the end of the spring bloom. We report substantial N2 fixation activities, reaching up to 65 nmol N L−1 d−1 and 1533 µmol N m−2 d−1 close to the Iberian Margin between 38.8° N and 40.7° N. Similar figures in the basin have only been reported in the temperate and tropical western North Atlantic waters with coastal, shelf or mesohaline characteristics, as opposed to the mostly open ocean conditions studied here. In agreement with previous studies, the qualitative assessment of nifH gene diversity (encoding the nitrogenase enzyme that fixes N2) suggested a predominance of heterotrophic diazotrophs, and the absence of filamentous cyanobacteria. At the sites where N2 fixation activity was highest sequences affiliated to UCYN-A1, obligate symbiont of eukaryotic prymnesiophyte algae, were recovered. The remaining phylotypes were non-cyanobacterial diazotrophs, known to live in association with suspended particles and zooplankton (i.e., Bacteroidetes, Firmicutes and Proteobacteria). Outside the area of exceptional activity, N2 fixation in the open ocean and at shelf-influenced sites was also relatively high, ranging from 81 to 384 µmol N m−2 d−1, but was undetectable in the central Bay of Biscay. We propose that the unexpectedly high heterotrophic N2 fixation activity recorded at the time of our study was sustained by the availability of phytoplankton derived organic matter (dissolved and/or particulate) resulting from the ongoing to post spring bloom. We pose that this organic material not only sustained bacterial production, but also provided sufficient nutrients essential for the nitrogenase activity (e.g., phosphorus). Dissolved Fe was supplied through atmospheric dust deposition during the month preceding our study and through advection of surface waters from the subtropical region and the shelf area. Our findings stress the need for a more detailed monitoring of the spatial and temporal distribution of oceanic N2 fixation in productive waters of the temperate North Atlantic to better constrain the basin-scale nitrogen input to the ocean inventory. [ABSTRACT FROM AUTHOR]