Estrogenic hormones, believed to exert most of their effects via the direct interaction of their receptors with chromatin, are found to increase cAMP in target breast cancer and uterine cells in culture and in the intact uterus in vivo. Increases in intracellular cAMP are evoked by very low concentrations of estradiol (half maximal at 10 pM) and by other physiologically active estrogens and antiestrogens, but not by an inactive estrogen stereoisomer. These increases in cAMP result from enhanced membrane adenylate cyclase activity by a mechanism that does not involve genomic actions of the hormones (are not blocked by inhibitors of RNA and protein synthesis). The estrogen-stimulated levels of cAMP are sufficient to activate transcription from cAMP response element-containing genes and reporter plasmid constructs. Our findings document a nongenomic action ofestrogenic hormones that involves the activation of an important second-messenger signaling system and suggest that estrogen regulation of cAMP may provide an additional mechanism by which this steroid hormone can alter the expression of genes.For many years, steroid hormones and peptide hormones have been considered to act via distinctly different mechanisms, the former via intracellular receptors acting through the genome (1, 2) and the latter via membrane-localized receptors that initially affect extranuclear activities, including the generation of second messengers such as cAMP. However, there has been increasing evidence for interactions between cAMP and estrogen in enhancing the growth of the mammary gland and breast cancer cells (3, 4) and for cAMP induction of estrogen-like uterine growth (5). As early as 1967, Szego and Davis (6) demonstrated a very rapid, acute elevation of uterine cAMP by estrogen treatment of rats in vivo that was confirmed in other reports (7,8), but several subsequent studies either failed to confirm this observation or reported only minimal effects that were considered to represent indirect effects of estrogen on cAMP mediated by estrogen-induced release of uterine epinephrine (9)(10)(11)(12). Recently, cAMP and other protein kinase activators have been documented to synergize with steroid hormone-occupied receptors, leading to enhanced steroid receptor-mediated transcription (13)(14)(15)(16)(17)(18), possibly by a mechanism involving phosphorylation of the receptor or associated transcription factors (14,(19)(20)(21).In this paper, we show that estrogen activates adenylate cyclase, markedly increasing the concentration of cAMP in estrogen-responsive breast cancer and uterine cells in culture and in the intact uterus of rats treated with estrogen in vivo, in a manner that does not require new RNA or protein synthesis. The intracellular concentrations of cAMP achieved by low, physiological levels of estrogen are substantial and sufficient to stimulate cAMP response element (CRE)-mediated gene transcription. Therefore, this nongenomic action of the steroid hormone estrogen involves the production of an important second mess...