The complexation reaction between Cu and four targeted endocrine-disrupting compounds (EDCs)-17beta-estradiol (E2), 17alpha-ethynylestradiol (EE2), 4-octylphenol (OP), and bisphenol A (BPA)-was studied using the fluorescence quenching method, with the changed EDC forms evaluated for their estrogenic activities using an E-screen assay. The results of the E-screen showed no estrogenic activity of Cu, whereas results with the EDCs indicated high cell proliferation (decreasing order of induction: E2, EE2, OP, and BPA). The results of the fluorescence quenching response and FTIR (Fourier transformation infrared) strongly support the formation of Cu complexes on the -OH functional group of the targeted EDCs when Cu was spiked at concentrations between 10(-7) and 10(-4) M. The degree of complexation was in the following order: E2, EE2, OP, and BPA. The E-screen result for three of the EDCs, with the exception of BPA, was similar when estimating the effects of Cu on the estrogenic activities of the EDCs, which were significantly reduced at alpha = 0.05 with increasing Cu concentration. Consequently, the results of the complexation reaction and E-screen assay with the Cu-EDC mixtures indicated that Cu plays a significant role in the assessment of the estrogenic activities of EDCs in natural waters.