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Aquaculture is a growing commercial activity worldwide, which resorts more and more often to microalgae as feed; the lipid composition of such microalgae is a critical factor with regard to the fish growth rate upon ingestion. The aim of this work was thus to study the influence of light intensity on the lipid profile of a known microalga, Pavlova lutheri. Several semi-continuous cultures were carried out, and biochemical parameters such as lipid, protein, carbohydrate, and chlorophyll contents were quantified. Lipids were specifically fractionated into classes by TLC, and those in each class were subjected to GC afterwards in an attempt to ascertain their fatty acid profile. Evidence was consequently provided which showed that cultures grown under low light intensity (9 W m⁻²) possess a higher fraction of eicosapentaenoic (EPA) and docosahexaenoic (DHA) acids esterified in polar classes—which are those with a more favorable role in aquaculture. It was also demonstrated that intermediate levels of light intensity (19 W m⁻²) may be misleading in terms of favorable effects upon EPA and DHA contents—because there is an increase in their total yields and productivities, but they appear mostly esterified into triacylglycerols; this may be a favorable deed for production and purification, but is metabolically not so effective in aquaculture. The highest EPA and DHA productivities attained were 1.29 and 0.69 mg L⁻¹ day⁻¹, respectively, at intermediate levels of light intensity (19 W m⁻²).