Estrogen rapidly activates MAPK in many cell types but the mechanisms have not been fully understood. We previously demonstrated that 17-beta-estradiol (estradiol) rapidly induced membrane translocation of estrogen receptor alpha (ERalpha) and activated MAPK in MCF-7 breast cancer cells. This study further determines the cause and effect relationship between the presence of membrane ERalpha and MAPK activation. ERalpha with a membrane localization signal (HE241G-mem) was expressed and compared with the ones in nucleus (HEGO) or cytosol (HE241G) localization. Confocal microscopy showed that HE241G-mem was expressed in the cell membrane as well as in the cytosol in COS-1 cells. HE241G localized in the cytosol and HEGO in the nucleus. Functional studies showed that only membrane ERalpha, not cytosol and nuclear ones, responded to estradiol by inducing MAPK phosphorylation. HE241G-mem neither increased basal nor estradiol-induced ERE promoter activation, indicating no transcriptional action involved. Our data support the view that membrane-associated ERalpha is critical in estrogen-initiated MAPK activation. (C) 2002 Elsevier Science (USA). All rights reserved.