The rapid, nongenomic effects of estrogen are increasingly recognized as playing an important role in several aspects of estrogen action. Rapid activation of the mitogen-activated protein kinase (MAPK) signaling pathway by estrogen is among the more recently identified of these effects. To explore the role of estrogen receptors (ERs) in mediating these effects, we have transfected ER-negative Rat-2 fibroblasts with complementary DNA clones encoding either human ERalpha or rat ERbeta and examined their ability to couple to activation of MAPK in response to 17beta-estradiol (17beta-E(2)) and other ligands. For both receptors, addition of E(2) resulted in a rapid phosphorylation of MAPK. Activation of MAPK in ERalpha-transfected cells was partially and completely blocked by the antiestrogens tamoxifen and ICI 182,780, respectively. In ERbeta-transfected cells, MAPK activation was less sensitive to inhibition by tamoxifen and ICI 182,780. We have also observed that, in this model system, a membrane-impermeable estrogen (BSA-E(2)) and 17alpha-E(2) were both able to activate MAPK in a manner similar to E(2) alone. Here also, ICI 182,780 blocked the ability of BSA-E(2) to activate MAPK through ERalpha, but failed to block ERbeta-mediated effects. BSA-E(2) treatment, however, failed to activate nuclear estrogen-response-element-mediated gene transcription. These data show that these nuclear ERs are necessary for estrogen's effects at the membrane. This model system will be useful in identifying molecular interactions involved in the rapid effects mediated by the ERs.