Wastewater reclamation and reuse is imperative in water-starved areas such as the southwestern United States. In the Tucson Active Management Area, a geographic region defined for the purpose of groundwater management, the total demand for water already exceeds the available water supply, which consists of renewable ground water and the regional entitlement to Central Arizona Project water. Thus, the regional demand cannot be satisfied without resorting to groundwater mining unless water is reclaimed for local beneficial use. Less certain are the acceptable uses for reclaimed water and the nature of use-dependent treatments or water quality requirements that will protect human and ecological health. Disruption of thyroid-mediated actions is among the possible risks from chronic exposure to environmental contaminants. Endocrine disrupting compounds are generally of greater concern in this context than other trace contaminants because of the very low concentrations at which hormones induce physiological responses. Accordingly, a sensitive nuclear-based bioassay system was developed in order to evaluate environmental samples. A luciferase-reporter construct and the human thyroid receptor β (TR-β) construct in the human hepatoma cell line (HepG2) and human medulloblastoma cells (TE671) was evaluated for sensitivity. The transfected cells were exposed to the thyroid hormone, T3, in order to establish a lower thyroid hormone detection limit for the new bioassay procedure. The assay was then applied to environmental samples containing organics concentrated from final effluent derived from a conventional secondary wastewater treatment plant. The effluent samples activated thyroid receptor-mediated transcription. Also in this study, a two-dimensional gel electrophoresis (2D-DIGE) was used as an in vitro bioassay to look for wastewater related alteration of cellular protein expression in the human breast cancer cell T47D. Steps in this bioassay include the one-dimensional separation of proteins by isoelectric focusing followed by orthogonal electrophoresis to isolate the proteins. The consistency of test response was examined for wastewater-dependent up- or down-regulation of protein expression. Some proteins that were upregulated were preliminarily identified via mass spectrometry. Here, the procedure is used to provide direct information regarding the probable effects of residual hormones in treated wastewater on the activities of human, estrogen-responsive cells in cell culture.