Reuse of municipal and industrial wastewater treatment plant (WWTP) effluent is an important component in augmenting global freshwater supplies. The Shenandoah River Watershed was selected to conduct on-site exposure experiments to assess endocrine disrupting characteristics of different source waters. This investigation of the Shenandoah River Watershed integrates WWTP wastewater reuse modeling, hydrological and chemical characterization, and in vivo endocrine disruption bioassessment to assess contaminant sources, exposure pathways, and biological effects. The percentage of accumulated WWTP effluent in each river reach (ACCWW) was used to predict environmental concentrations for consumer product chemicals (boron), pharmaceutical compounds (carbamazepine), and steroidal estrogens (estrone, 17-beta-estradiol, estriol, and 17-alpha-ethinylestradiol). Fish endocrine disruption was evaluated using vitellogenin induction in male or juvenile fathead minnows. Water samples were analyzed for >500 inorganic and organic constituents to characterize the complex contaminant mixtures. Municipal ACCWW at drinking water treatment plant surface-water intakes ranged from <0.01 to 2.1 % under mean-annual streamflow and up to 4.7 % under August streamflow. Measured and predicted environmental concentrations resulted in 17-beta-estradiol equivalency quotients ranging from <0.05 to 5.1 ng L-1 indicating low-to-moderate risk of fish endocrine disruption. Results from the fish exposure experiments also showed limited estrogenic effects as indicated by the low (0.5- to 3.2-fold) vitellogenin induction.