Systemic administration of the ovarian hormone estrogen to castrated female rats improves performance during acquisition on a variety of measures of spatial working memory, including the radial arm maze. Systemic estrogen treatments also prevent performance impairments induced by the systemic administration of scopolamine, a compound that blocks muscarinic receptors for the neurotransmitter acetylcholine. In addition, systemic estrogen treatments prevent impairments in spatial working memory caused by the direct infusion of scopolamine into the hippocampal formation Performance enhancements during acquisition, as well as prevention of scopolamine-induced impairments by estrogen treatments were localized to specific brain regions in this series of experiments. Results of the current study indicate that when placed unilaterally into the medial septal area, cannulae containing mixtures of estradiol:cholesterol in concentrations of either 1:125 or 1:250 improved performance during acquisition of an 8-arm baited radial maze task with 1-hour delays interposed between the fourth and fifth arm choices compared to implants containing pure cholesterol. Implants containing estradiol that were placed bilaterally into either the hippocampus, or a cortical region dorsal to the hippocampus were ineffective. These results indicate that the medial septum is a site at which estrogen may act to improve performance during acquisition of spatial working memory tasks. In a second segment of the present study, the site at which estrogen acts to prevent performance impairments induced by systemic administration of scopolamine was investigated. On a standard version of the radial-arm maze task in which delays were not placed between the fourth and fifth arm choices, scopolamine impaired performance of females with implants containing either estrogen or cholesterol in either the medial septum or cerebral cortex. In contrast, animals with hippocampal estradiol implants at concentrations of either 1:1, 1:125, or 1:250 did not display similar impairments following scopolamine injection, indicating that estrogen may act at the hippocampus, but not medial septum or cerebral cortex to prevent the deficits in performance caused by scopolamine. These results indicate that the effects of estrogen on learning and memory are central in nature, and demonstrate that the hippocampus and medial septum are critically involved in these processes