A novel yeast cell-based assay was developed for the detection of estrogenic activity in wastewater. Recombinant Arxula adeninivorans strains were engineered to co-express the human estrogen receptor alpha (hERalpha) and a Klebsiella-derived phytase (phyK) reporter gene under the control of an A. adeninivorans-derived glucoamylase (GAA) promoter which had been modified by the insertion of estrogen-responsive elements (EREs). In the presence of estrogenic compounds, hERalpha dimerizes and binds to the estrogen. Reporter gene expression is induced by subsequent binding of the hERalpha-dimer/estrogen complex to estrogen responsive elements (ERE) in the promoter. The insertion of different numbers of EREs in three alternative promoter positions and its effect on reporter gene expression were assessed. In one of the constructs, a detection limit of 5 ng l(-1) and a determination limit of 10 ng l(-1) for 17beta-estradiol-like activity was achieved. The photometric assay used enabled estrogen determination in sewage samples within 30 h.