In MCF-7 cells, estradiol (E2) and pure antiestrogens (AEs) decrease estrogen receptor alpha (ER) levels, while AEs with partial estrogenic activity lead to ER alpha accumulation. Using immunocytochemistry, we found that cells pre-exposed to one of such ligands, when plated with untreated cells, led to similar ER changes in the latter. Conditioned media (CMs) prepared from stimulated cells displayed identical regulatory effects even after strong dilution; they also modulated ERE-dependent transcriptional activity. Evaluation of residual ligand concentrations in CMs rejected the possibility of a major interference of the former. Cycloheximide, which inhibits E2-induced down-regulation, failed to block the influence of CM(E2) in agreement with this view. DCC-treatment of CMs abrogated their effects, suggesting the release of hydrophobic compound(s) which regulate ER and/or amplify the effect of extremely low amounts of residual ligands. Such a release appears independent of ER since CMs from MDA-MB-231 cells (ER-negative) were effective as their autologous media on MCF-7 cells.