This paper describes the screening of 22 extracts from 18 different aquatic environmental samples for androgenic activity, including indirect and interactive effects on androgen receptor (AR)-mediated signal transduction, using the AR-LUX bioassay. Four samples, originating from an industrial wastewater treatment plant (WTP) or the river Meuse, were shown to contain substantial androgenic activity. Moreover, the samples originating from the industrial WTP showed an enhancement of the maximal androgenic response relative to that elicited by the standard androgen methyltrienolone (R1881) in the AR-LUX assay. This indicates the involvement of cellular mechanisms other than receptor-ligand interaction influencing AR-regulated pathways. This also demonstrates the additional value of cell based assays featuring a more complete array of fully functional interacting pathways. Chemical analysis using GC-MS confirmed the presence of a number of androgens and also estrogens in these WTP samples. Subsequently, we showed that estrone and tributyltin hydride (TBT-H) enhance the response to androgens. This indicates that the presence of numerous compounds in addition to androgens in environmental mixtures might very well result in a more profound perturbation of the normal physiology of exposed organisms than estimated based on the androgen levels alone. Therefore, risk assessment of environmental samples should include an evaluation of the presence and the interactive effects of (ant)agonists of carefully selected relevant cellular receptors in order to provide a realistic estimate of the integrated ecotoxicological risk of the compounds present. © 2004 Elsevier B.V. All rights reserved.