The three-spined stickleback (Gasterosteus aculeatus) has quantifiable biomarkers of exposure to estrogens (vitellogenin), androgens (spiggin) and aryl hydrocarbon receptor (AhR) agonists (EROD activity) and is therefore a promising test species for biomonitoring of reprotoxic chemicals in aquatic environments. In this study we evaluated the effects of 17alpha-ethynylestradiol (EE(2)) on EROD activity, induction of vitellogenin and spiggin, hepatosomatic index (HSI), ovarian somatic index (OSI) and nephrosomatic index (NSI). Adult male and female three-spined sticklebacks were exposed to concentrations of 0-170 ng EE(2)/l (measured concentrations) in a flow-through system for 21 days. Exposure to 170 ng EE(2)/l resulted in a significant 8- and 9-fold induction of gill EROD activity in males and females, respectively. In livers, EROD activity expressed in relation to microsomal protein content was suppressed due to a significant increase in microsomal protein content. Hepatic EROD activity per se expressed as picomol/min was not affected by exposure to EE(2). The lowest observed effect concentration for induction of vitellogenin in males was 53.7 ng EE(2)/l. In females, vitellogenin levels were significantly higher in those exposed to 170 ng EE(2)/l compared to controls. Spiggin production was significantly inhibited and NSI lower in males exposed to 170 ng EE(2)/l. In both females and males LSI was significantly higher in fish exposed to 170 ng EE(2)/l than in controls. In females exposed to 170 ng EE(2)/l, OSI was significantly lower and NSI higher than controls. The observed results from this study show that a synthetic estrogen can affect the well-known biomarker of exposure for dioxin-like compounds, EROD activity, and further that this response can differ between tissues. These findings are important for interpretation of biomonitoring data.