A novel and efficient analytical methodology is proposed for extracting and preconcentrating three kinds of estrogens (17 beta-estradiol (beta E2), estrone (E1), and diethylstilbestrol (DES)) in human urine prior to high-performance liquid chromatography (HPLC) analysis. It is based on the induction of micellar organized medium by using a nonionic surfactant (Tergitol TMN-6) to extract the target estrogens. Ultrasound was applied to enhance the extraction efficiency. Parameters affecting the extraction of target analytes including the concentration of surfactant, temperature, extraction time, sample pH, ionic strength, and centrifuging time were investigated. Under the optimum conditions, the linear range of beta E2, E1, and DES was from 5.0 to 1000 ng/ml. All correlation coefficients of the calibration curves were higher than 0.997. The relative standard deviations (RSD, n = 5) were 2.36-5.27% and the limits of detection (LOD) were 0.1, 0.2, and 0.1 ng/ml for beta E2, E1, and DES in human urine, respectively. The results indicated that the method was successfully applied for analyzing beta E2, E1, and DES in human urine. (C) 2011 Elsevier Inc. All rights reserved.