The application of bioassays to assess the occurrence of estrogenic compounds in the environment is increasing in both a scientific and statutory context. The availability of appropriate validated methods for sample pre-treatment and analysis is crucial for the successful implementation of bioassays. Here, we present a sample preparation method for the bioassay screening of estrogenic activity in sediment with the in vitro Estrogen Receptor mediated Chemical Activated LUciferase gene eXpression (ER-CALUX) assay. The method makes use of an Accelerated Solvent (ASE) or Soxhlet extraction with a mixture of dichloromethane and acetone (3:1, v/v), followed by clean up of the extract by Gel Permeation Chromatography (GPC). Recoveries of a panel of 17 pollutants differing largely in physical-chemical properties from spiked sediment were determined and appeared to be on average about 86%. Furthermore, the estrogenic potencies of all test compounds were individually assessed by determination of concentration-response relationships in the ER-CALUX assay. Concentration dependent estrogenic potency was found for 14 of the 17 compounds, with potencies of about 10(5) to 10(7) fold lower than the natural estrogenic hormone 17beta-estradiol. Anti-estrogenic potency was assessed by testing combinations of estradiol and individual test compounds, but was found for none of the compounds. The low estrogenic activity of the test compounds in the spiking mixture was well recovered during GPC treatment of the pure mixture, but did not contribute significantly to the background estrogenic activity present in the spiked sediment. Application of the method to field samples showed that estrogenic activity can be found at different types of locations, and demonstrated that levels between locations may vary considerably over relatively short distances.