The nutritional value of diatoms for grazers has been frequently addressed in experiments. However, little is known about the effects of culture conditions on the quality of algae as food for higher trophic levels. We examined the fatty acid composition of four different strains of the diatom Thalassiosira rotula under nitrate- and phosphate-limited conditions, in natural seawater, at different light intensities, in different growth phases, and with addition of low doses of octadienal. The same samples were tested for the production of fatty acid-derived polyunsaturated aldehydes (PUAs: heptadienal, octadienal, octatrienal, decadienal, decatrienal) that are reported to have negative effects on the hatching and survival of copepod offspring. Phosphate- and nitrate-limited cultures as well as those in the exponential growth phase under phosphate and nitrate-replete conditions contained less eicosapentaenoic acid (EPA) and produced less PUA than algae cultured in non-limiting F/2 medium in the stationary and decline phases. Light intensity had no significant influence on either fatty acid composition or PUA production. We further show that addition of octadienal in methanol (0.02 μM every second day) increased the potential wound-activated PUA production in the CCMP 1018 strain. Our findings also indicate that the production of decatrienal, the major PUA in T. rotula, was related to the cell content of EPA.