The aim of this study was to identify and characterize an alternative pathway through which environmental estrogenic compounds may mediate their intracellular effects. Three human breast cancer cell lines were employed including MCF-7 cells, which express both ERalpha and ERbeta; MDA-MB-231 cells, which express ERbeta but not ERalpha; and SKBR-3 cells, which express neither ERalpha nor ERbeta. The effect of environmental estrogenic compounds on intracellular calcium ion concentration ([Ca2+](i)) was measured and compared to that of 17beta-estradiol (E2). A rapid and maintained increase in [Ca2+](i) was observed following the application of nanomolar concentrations of environmental estrogens and E2 regardless of the expression of ERalpha and ERbeta. Removal of extracellular Ca2+ completely abolished the steroid-induced [Ca2+](i) increase. Pre-treatment of cells with the estrogen receptor (ER) antagonist ICI 182,780 had no effect on either basal [Ca2+](i) or the steroid-triggered [Ca2+](i) response.In summary, we have demonstrated ER independent rapid non-genomic effects of environmental estrogenic compounds, at nanomolar concentrations. on [Ca2+](i). The results of this study demonstrate an alternative pathway to explain potent intracellular effects of endocrine disrupting chemicals. (C) 2004 Elsevier Ireland Ltd. All rights reserved.