We have investigated the response dynamics of the estrogen-dependent genes vitellogenin (VTG) and the vitelline envelope proteins (VEPs) as well as circulating VTG in immature female rainbow trout (Oncorhynchus mykiss) exposed to 17β-estradiol (E2) and 17α-efhinylestradiol (EE2) for periods of 7 and 14 d. Gene responses were quantified by measurement of messenger RNA (mRNA) in liver extracts using a chemiluminescent hybridization protection assay. Circulating VTG was measured by a homologous enzyme-linked immunosorbent assay. Exposure to both E2 and EE2 induced concentration-dependent increases in all biomarkers. The data presented indicate that VEP genes may be more sensitive to estrogens than the VTG gene. The biomarker lowest-observed-effect concentrations (biomarkerLOEC) in the 14-d study with E2 were 14 ng/L (VTG protein, VTG mRNA, VEPβ, and VEPγ) or 4.8 ng/L (VEPα). The EE2 was 5- to 66-fold more potent depending on the biomarker studied. In the 7-d study, all biomarkers were elevated after 48-h exposure to E2, with biomarkerLOECs of 30 ng/L (VTG protein, VTG mRNA, and VEPγ) or 9.7 ng/L (VEPα and VEPβ). Vitellogenin mRNA was induced up to 1,000-fold above baseline, and this translated into an increase of approximately 50,000-fold in circulating VTG. In conclusion, all biomarkers responded to estrogen exposure at environmentally relevant concentrations.