Crude oil is a complex mixture of compounds of which the water-soluble fraction (WSF) is considered to be bioavailable and potentially toxic to aquatic biota. Containing numerous compounds, WSF becomes a source of multiple chemical stressors to wildlife when introduced into the environment. To study the combined effects of WSF components on aquatic biota, the model species zebrafish (Danio rerio Hamilton) was exposed for 24 or 72 h to 10 or 50% WSF solution of known composition, generated from artificially weathered North Sea crude oil. Hepatic expression of genes involved in the aryl hydrocarbon receptor-cytochrome P-450 1A (AhR-CYP1A) pathway (AhR2, AhRR1, CYP1A1) and steroidogenesis (StAR, CYP11A, 3β-HSD, CYP19A, CYP19B) was measured, as well as estrogen receptors ERα and ERβ1. Induction of CYP1A and particularly of AhRR1 was observed while ERα and steroidogenic enzymes CYP11A and 3β-HSD were downregulated. Regression analysis demonstrated a negative relationship between AhR-CYP1A pathway and endocrine transcript levels, although causality remains to be established. These findings indicate that exposure to WSF of oil disrupts steroidogenesis and may therefore constitute a potential risk for reproductive ability of aquatic organisms. In addition, it is proposed that hepatic gene expression of AhRR1 may serve as a novel biomarker of WSF exposure.