Wastewater treatment plant effluents contain a variety of endocrine disrupting chemicals (EDCs), including chemicals with estrogenic activity such as 17β-estradiol (E2), 17α-ethinyl estradiol (EE2), and nonylphenols. These substances can affect both behavior and physiology in vertebrate animals. To explore the presence and effects of these EDCs in a natural setting, juvenile and adult male fathead minnows, Pimephales promelas, were held in cages upstream and downstream of the effluent site of a wastewater treatment plant for 21 days and subsequently tested for changes in reproductive behaviors and production of vitellogenin. Additionally, estrogenic activity in the stream was measured using a yeast bioassay. Estrogenicity was found to be significantly higher downstream of the wastewater effluent when compared to levels upstream. Vitellogenin levels did not show a correlational pattern with levels of estrogenicity in the water, but two measures of reproductive behaviors occurred significantly less often in downstream males than upstream males. This suggests that a brief (three-week) exposure to stream water containing wastewater treatment plant effluent can bring about changes in reproductive behavior of fish and that behavior may be more sensitive to low levels of environmental endocrine disruptors than vitellogenin production.