Organisms at the base of aquatic food webs synthesize essential nutrients, such as omega-3 polyunsaturated fatty acids (n-3 PUFA), which are transferred to consumers at higher trophic levels. Many consumers, requiring n-3 long-chain (LC) PUFA, such as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), have limited ability to bioconvert them from the essential dietary precursor α-linolenic acid (ALA) and thus rely on dietary provision of LC-PUFA. We investigated LC-PUFA metabolism in freshwater zooplankton using stable hydrogen isotopes (d²H) of fatty acids as tracers. We conducted feeding experiments with the freshwater keystone grazer Daphnia to quantify changes in the d²H value of body FA in response to the FA composition of their food and the d²H value of the ambient water. The isotopic composition of LC-PUFA changed in Daphnia, depending on the integration of ²H from ambient water during de novo synthesis or bioconversion from dietary precursors, allowing us to distinguish dietary from bioconverted EPA in body tissue. We tested the applicability of these laboratory findings in a field setting by analyzing d²H values of PUFA in primary producers and consumers in eutrophic ponds to track EPA sources of zooplankton. Multilinear regression models that included conversion of ALA to EPA correlated better with zooplankton d²H_EPA than seston d²H_EPA at low dietary EPA supply. This study provides evidence that zooplankton can compensate for low dietary EPA supply by activating LC-PUFA biosynthesis and shows that herbivorous zooplankton play a crucial role in upgrading FA for higher trophic levels during low dietary EPA supply.