Culture studies of microorganisms have shown that the hydrogen isotopic composition of fatty acids depends on their metabolism, but there are only few environmental studies available to confirm this observation. Here we studied the seasonal variability of the deuterium/hydrogen (D/H) ratio of fatty acids in the coastal Dutch North Sea and compared this with the diversity of the phyto- and bacterioplankton. Over the year, the stable hydrogen isotopic fractionation factor ε between fatty acids and water ranged between −172 ‰ and −237 ‰, the algal-derived polyunsaturated fatty acid n C20:5 being the most D-depleted and n C18:0 the least D-depleted fatty acid. The D-depleted n C20:5 is in agreement with culture studies, which indicates that photoautotrophic microorganisms produce fatty acids which are significantly depleted in D relative to water. The ε lipid/water of all fatty acids showed a transient shift towards increased fractionation during the spring phytoplankton bloom, indicated by increasing chlorophyll a concentrations and relative abundance of the n C20:5 PUFA, suggesting increased contributions of photoautotrophy. Time periods with decreased fractionation (less negative ε lipid/water values) can be explained by an increased contribution by heterotrophy to the fatty acid pool. Our results show that the hydrogen isotopic composition of fatty acids is a useful tool to assess the community metabolism of coastal plankton.