In February 2003 the U.S. EPA published a final rule on National Pollutant Discharge Elimination System Permit Regulation and Effluent Limitation Guidelines and Standards for Concentrated Animal Feeding Operations (CAFOs). Manure and wastewater from CAFOs have the potential to contribute pollutants such as nitrogen and phosphorus, organic matter, sediments, pathogens, heavy metals, hormones, antibiotics, and ammonia to the environment. Excess nutrients in water (i.e., nitrogen and phosphorus) can result in or contribute to low levels of dissolved oxygen (anoxia), eutrophication, and toxic algal blooms. The CAFO rule mainly covers the control of nutrients and bacterial contamination. This report investigated the potential ecological effects of hormonally active substances associated with discharges from cattle-feeding operations. Revalor S7 implants, which contain both trenbolone acetate and 17$-estradiol were used on the 6000-plus cattle that were housed at the study CAFO facility. Trenbolone acetate is a synthetic anabolic steroid that is widely used in beef production in the US. Metabolites of trenbolone acetate include the stereoisomers 17 inch - and 17$-trenbolone, both of which are stable in animal wastes and are relatively potent androgens in reproduction assays with the fathead minnow, laboratory rat and other species. The purpose of this study was to evaluate the potential of a fathead minnow vitellogenin gene expression assay as an indicator of androgenic substances like 17 inch - and 17$-trenbolone in a beef feedlot discharge, and in river water upstream and downstream from the discharge. Our objective was to see if sexually mature female fathead minnows would show reduced levels of vitellogenin gene expression when exposed to androgens. Samples were collected on three different occasions during 2002 and 2003. No consistent changes in vitellogenin gene expression were detected in female fathead minnows exposed to samples collected from a CAFO drain or downstream stream samples that had detectable concentrations of Trenbolone shown in laboratory studies to cause effects on reproduction.