In this comparative study, the suitability of the commonly used in vivo biomarker for estrogenicity, vitellogenin (VTG), upon waterborne exposure to known environmental estrogens is evaluated in both male zebrafish (Danio rerio) and juvenile rainbow trout (Oncorhynchus mykiss). The results from initial experiments in which both species were exposed to 4-tert-octylphenol (OP) or 17 alpha-ethynylestradiol under semistatic conditions for 3 weeks demonstrated a difference in species sensitivity for OP exposure. Additional dose-response studies (semistatic, 3 weeks) with 4-nonylphenol (20, 100, and 500 microg/L), bisphenol A (40, 200, and 1000 microg/L), dibutylphthalate (40, 200, and 1000 microg/L DBP), and 17beta-estradiol (20 and 100 ng/L E2) were conducted. All these compounds, except for DBP, were found to be estrogenic to both fish species. The results demonstrated a difference in species sensitivity for NP with the zebrafish being about 5 times less sensitive. For the other compounds tested, no indications for a difference in species sensitivity was found. The results from this study demonstrated that both fish species can be used for the detection of VTG as biomarker for estrogenicity, taken into the potential interspecies differences in sensitivity which might be important for the evaluation of fish population effects.