17-estradiol greatly and dose-dependently stimulates [³H]arachidonic acid release from the human amnion-like WISH cells. This action is abolished by the phospholipase A₂ inhibitor AACOCF₃, significantly reduced by the estrogen receptor antagonist ICI 182,780, and uninfluenced by cycloheximide. The estradiol-BSA conjugate E₂coBSA, which binds putative membrane estrogen receptors and is unable to enter the cell, also highly stimulates [³H]arachidonic acid release from WISH cells, even if to a lesser extent compared with 17-estradiol. The fluorescent conjugate E₂coBSA-FITC specifically binds to the surface of a subset of intact WISH cells, and labeling intensity appears dose- and time-dependent. Cell permeabilization results in a dense intracellular staining, mainly in the peripheral cytoplasm. H-150, an antibody against the N terminus of human ER, also labels the plasma membrane of intact WISH cells and the cytoplasm of permeabilized cells. Almost no labeling is observed using ER-21, an antibody against the N terminus of human ER. RT-PCR evidences the presence of mRNA for ER, not for ER. Our data suggest that 17-estradiol stimulates [³H]arachidonic acid release from WISH cells through an apparently non-genomic pathway and interacting with membrane binding sites. These last are, at least in part, similar if not identical to ER.