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The cyanobacterium Raphidiopsis raciborskii is of environmental and social concern in view of its toxicity, bloom-forming characteristics and increasingly widespread occurrence. However, while availability of macronutrients and micronutrients such as N and Fe are critically important for the growth and metabolism of this organism, the physiological response of toxic and non-toxic strains of R. raciborskii to varying Fe and N availabilities remains unclear. By determining physiological parameters as a function of Fe and N availability, we demonstrate that R. raciborskii growth and N 2 -fixing activity are facilitated at higher Fe availability under N 2 -limited conditions with faster growth of the CS-506 (cylindrospermopsin-producing) strain compared with that of CS-509 (the non-toxic) strain. Radiolabelled Fe uptake assays indicated that R. raciborskii acclimated under Fe-limited conditions acquires Fe at significantly higher rates than under Fe replete conditions, principally via unchelated Fe(II) generated as a result of photoreduction of complexed Fe(III). While N 2 -fixation of both strains occurred during both day and night, the CS-506 strain overall exhibited higher N 2 -fixing and Fe uptake rates than the CS-509 strain under N-deficient and Fe-limited conditions. The findings of this study highlight that Fe availability is of significance for the ecological advantage of CS-506 over CS-509 in N-deficient freshwaters., (© 2019 Society for Applied Microbiology and John Wiley & Sons Ltd.)