In this study, the E-SCREEN assay was optimized and validated for the sensitive quantitative determination of the total estrogenicity in river samples. River water and sediment samples were collected and analyzed with the E-SCREEN. River water (10 l) was extracted using combined solid-phase extraction in static adsorption mode with Soxhlet extraction. Estrogenic pollutants adsorbed to the XAD-4 resin were recovered with 98.24 +/- 5.90% efficiency by elution with ethyl acetate and dichloromethane (1:9). The detection limit by 17beta-estradiol equivalent concentration (EEQ) of the E-SCREEN assay was 8.03 pg EEQ/l. Among the water samples, the estrogenic activity was observed to be higher downstream of the Kumho river (7.43 ng EEQ/l) and upstream of Kum river (2.05 ng EEQ/l) than in other samples. More than 3 mg of equivalent sediment samples from the Kumho river, Kum river and Miho stream showed partial agonistic effects, and the Mankyung river showed a partial agonistic effect with only 1.5 mg of sediment. The highest value of RPE was 83.34 downstream of the Kumho river, and the lowest value of RPE was 6.52 downstream of the Miho stream. Full estrogen agonistic activities were observed downstream of the Kumho river and upstream of the Kum river. The partial agonistic activity was observed in upstream of the Kumho river, downstream of the Mankyung river, and upstream of the Miho stream, and no agonistic action was observed downstream of the Kum river or Miho stream, or upstream of the Mankyung river. The total estrogenic activity in the river water and sediment samples was between 0.50 pg/L and 7.4 ng/L, 3.39 pg/g and 10.70 pg/g.