Nuclear magnetic resonance (NMR) based metabolomics in combination with multivariate data analysis may become valuable tools to study environmental effects of pharmaceuticals and other chemicals in aquatic organisms. To explore the usefulness of this approach in fish, we have used 1H NMR metabolomics to compare blood plasma and plasma lipid extracts from rainbow trout exposed to the synthetic contraceptive estrogen ethinylestradiol (EE2) with plasma from control fish. The plasma metabolite profile was affected in fish exposed to 10 ng/L but not 0.87 ng/L of EE2, which was in agreement with an induced vitellogenin synthesis in the high dose group only, as measured by ELISA. The main affected metabolites were vitellogenin, alanine, phospholipids and cholesterol. The responses identified by this discovery-driven method could be put in context with previous knowledge of the effects of estrogens on fish. This adds confidence to the approach of using NMR metabolomics to identify environmental effects of pharmaceuticals and other contaminants.