In recent years, concerns have been raised that low concentrations of chemicals may alter the normal functions of the endocrine system, resulting in potentially significant adverse effects on growth, reproduction, and/or development. For domestic wastewater discharges to surface water bodies, estrogenic activity of effluents has been suggested by chemical analysis, "biomarkers", and /or in vitro assays. Chemical Measurements. Using sensitive analytical techniques, natural or synthetic hormones and chemicals that mimic estrogen, have been detected in some domestic wastewater effluents and associated watersheds at low concentrations. (e.g. USGS Reconnaissance, Kolpin et al, 2002) Biomarkers. Several studies have demonstrated physiological changes in fish that may be attributed to exposure to estrogenic compounds. These include the presence of vitellogenin protein in male fish and/or intersex conditions. In vitro assays. The Yeast Estrogen Screen (YES) has been used to characterize estrogenicity of effluents, influents, biosolids and surface waters. This two-year WERF project explored approaches to evaluate the potential for biomarker formation as a result of effluent exposures, and the subsequent relevance of the emerging assays and physiological measurements on potential adverse impacts to individuals or populations of fish in the receiving streams. The endocrine system is complex, and many factors can influence the physiological measurements, including methods, sex, age, reproductive status, seasonal and circadian rhythms, diet, temperature, etc. and produce transient changes in physiology but no significant effect on the individual. This study highlights issues in the measurement and interpretation of biomarkers based on laboratory and field studies. Key observations made during this study include the following: Exposures to estradiol (E2) above the threshold level of approximately 0.1 nM (27 ng/L) can produce detectable plasma vitellogenin in male fathead minnows in less than a week. Data suggest a potential seasonal influence on this response. Vitellogenin will be detectable in juvenile trout at lower estrogen concentrations. Transient levels of estrogenic compounds in effluents above these levels could result in accumulation of protein. A short-term reproductive test was performed with E2 and an anti-estrogen to evaluate the presence of vitellogenin with reproductive success. Vitellogenin levels were elevated in the presence of E2, but a significant decrease in egg production was observed only in the presence of the anti-estrogen. This 21 day exposure was done with an E2 concentration of 0.5 nM, higher than typical levels in effluents.