Hydrogen isotope ratios of biomarkers have been shown to reflect water isotope ratios, and in some cases correlate significantly with salinity. The δ2H-salinity relationship is best studied for long-chain alkenones, biomarkers for haptophyte algae, and is known to be influenced by a number of different environmental parameters. It is not fully known why δ2H ratios of lipids retain a correlation to salinity, and whether this is a general feature for other lipids produced by haptophyte algae. Here, we analyzed δ2H ratios of three fatty acids, brassicasterol, long-chain C37 alkenones and phytol from three different haptophyte species grown over a range of salinities. Lipids synthesized in the cytosol, or relying on precursors of cytosolic origin, show a correlation between their δ2H ratios and salinity. In contrast, biosynthesis in the chloroplast, or utilizing precursors created in the chloroplast, yields lipids that do not show a correlation between δ2H ratios and salinity. This leads to the conclusion that location of metabolism is the first-order control on the salinity signal retained in δ2H ratios of certain lipids. Additionally, we found that δ2H ratios of alkenones from batch cultures of the Group II haptophyte species Tisochrysis lutea correlate positively with temperature, contrary to findings from cultures of Group III haptophytes, but retain a similar response to nutrient availability in line with other Group III haptophytes.