Among micro- and macroalgae, the mechanisms of nitrate uptake, storage, and reduction are central to questions in modern nitrogen cycling, paleoceanography, and algal cultivation. These mechanisms can be investigated by studying nitrogen isotope fractionation during nitrate assimilation, through isotopic measurements of external and intracellular nitrate as well as biomass. Previous studies proposed a one-intracellular-nitrate-pool framework that predicts an inverse relationship between the organism-level isotope effect and the isotopic difference between external and internal nitrate. In continuous cultures of the marine diatom Thalassiosira weissflogii and two red macroalgae, Eucheuma perplexum and Sarcodia suae, we observe a correlation between the organism-level isotope effect of nitrate assimilation and nitrate efflux-to-uptake ratio that is broadly consistent with previous studies. However, across environmental conditions, the observed organism-level isotope effects and/or the intracellular nitrate ¹⁵N and ¹⁸O enrichments are lower than predicted by the original one-internal-pool framework. These deviations require a contribution from low-δ¹⁵N and -δ¹⁸O intracellular nitrate that is not directly subjected to fractionation by the nitrate reductase. Calculations with a time-dependent multi-box model show that at least two internal pools are needed to explain observations, implying spatial or temporal separation between nitrate storage and reduction. Model results provide constraints on the cellular structure of internal nitrate pools in micro- and macroalgae, offering insight into the strategies algae use to store nitrate and maximize their growth in the face of varying nitrate availability. 

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