Two haptophyte algae, Emiliania huxleyi and Gephyrocapsa oceanica, were cultured at different temperatures and salinities to investigate the impact of these factors on the hydrogen isotopic composition of long chain alkenones synthesized by these algae. Results showed that alkenones synthesized by G. oceanica were on average depleted in D by 30 compared to those of E. huxleyi when grown under similar temperature and salinity conditions. The fractionation factor, αalkenones-H2O, ranged from 0.760 to 0.815 for E. huxleyi and from 0.741 to 0.788 for G. oceanica. There was no significant correlation of αalkenones-H2O with temperature but a positive linear correlation was observed between αalkenones-H2O and salinity with ~3 change in fractionation per salinity unit and a negative correlation between αalkenones-H2O and growth rate. This suggests that both salinity and growth rate can have a substantial impact on the stable hydrogen isotopic composition of long chain alkenones in natural environments.