Zhang and Sachs [Hydrogen isotope fractionation in freshwater algae: I. Variations among lipids and species. Organic Geochemistry 38, 582–608, 2007] demonstrated that algal lipid δD values track water δD values with high fidelity ( R 2 > 0.99), but that D/ H fractionation varied among lipids and algal species. Here we report on the influence of temperature and nitrogen limitation on D/ H fractionation in lipids from cultured microalgae. Two species of freshwater green algae, Eudorina unicocca and Volvox aureus, were grown in batch culture at 15 °C and 25 °C. Increased D/ H fractionation of 2−4‰/°C occurred at the higher temperature in all lipids analyzed. The marine diatom Thalassiosira pseudonana was grown in continuous culture under nitrogen replete (NR) and nitrogen limited (NL) conditions, resulting in a growth rate that was 4.4 fold lower under the latter conditions. The fatty acid content of NL cells was approximately 4 fold higher than in NR cells, whereas the sterol content was similar in both. While sterols from the NL culture were enriched in deuterium by 37‰ relative to the NR culture, fatty acids from both cultures had similar δD values, implying that D/ H fractionation during isoprenoid (branched) lipid synthesis is affected by nitrogen limitation, but D/ H fractionation during acetogenic (linear) lipid synthesis is not. Cross-talk of the precursor isopentenyl diphosphate between the cytosolic MVA and plastidic DOXP/MEP synthetic pathways is a plausible mechanism for the observed D/ H differences between isoprenoid and acetogenic lipids. This preliminary study highlights the need to consider both the type of lipid and potential changes in growth conditions in paleoenvironmental studies using lipid D/ H ratios.