Estrogens regulate the formation of excitatory synaptic connections in the hippocampus of female rats. Because the adult hippocampus has a very low concentration of intracellular estrogen receptors, it is unclear whether a conventional genomic mechanism is involved. Nonsteroidal estrogen antagonists are useful tools to study estrogen action because they can provide pharmacological data in favor of a particular pathway of estrogen action and evidence against other pathways. To investigate the role of intracellular estrogen receptors in the estrogen induction of synapse formation, we took advantage of previous studies in which we had shown that an estrogen antagonist, CI-628, enters the brain and blocks estrogen induction of progestin receptors to study whether the same antagonist would either mimic or block effects of estradiol to induce excitatory spine synapses. Using silver impregnation of neurons by the single section Golgi technique and morphometric analysis, we found that CI-628 effectively prevented estrogen induction of spines on CA1 pyramidal neurons, without having any agonist effects of its own. This result is consistent with an action of estradiol via intracellular estrogen receptors that are known to be expressed by interneurons within the hippocampus.