This study is aiming at examining the conditions of a testing method for comprehensive environmental-like activity in wastewater using recombinant yeast. Using this method, 43 chemicals that are suspected to have the estrogen-like effects measured. Then, their estrogen-like activities were evaluated as the 17 β-estradiol equivalent by comparison with the assay results of 17β-estradiol. 17β-estradiol equivalent of influent and effluent sampled from 10 wastewater treatment plants in summer and autumn were measured with this method. The endocrine disruptors (EDs) concentrations in the wastewater were calculated into the theoretical17 β-estradiol equivalents based on the chemical evaluation. The results suggest that w astewater treatment plants effectively reduce estrogen-like substances during treatment, and that there weresome differences between 17β-estradiol equivalent with the recombinant yeast and the theoretical 17β-estradiol equivalent based on the chemical evaluation. Therefore, it is implied that unknown estrogen-like substances or antagonists might exist in wastewater.