Actions of estrogen include mechanisms leading to alterations in gene transcription that may be independent of nuclear estrogen receptors, as well as those involving direct action of the estrogen receptor on the genome. Also, the influence of estrogen in the brain appears to extend well beyond areas associated with reproduction and may include forebrain areas linked to affective and cognitive behaviors. We investigated the effects of acute and long-term estradiol benzoate (E2) treatment on total and phosphorylated cyclic AMP responsive element-binding (CREB) protein levels and on cyclic AMP response element (CRE)-DNA binding in forebrain areas of ovariectomized (OVX) rats. Long-term E2 treatment increased CRE-DNA binding in the amygdala but not in hippocampus, frontal cortex, or cerebellum. The increase in CRE-DNA binding in the amygdala was associated with increased levels of total and phosphorylated CREB (pCREB) protein during protracted E2 exposure. To localize the estrogenic effect in the amygdala and determine if an effect in one hippocampal region was masked by a lack of effect in another subregion, we performed immunolabeling of pCREB in brain structures of chronically treated OVX animals with or without E2. This treatment resulted in a significant increase in relative total immunolabeled nuclei in the anteroventral subdivision of the medial amygdala. In the hippocampus, a significant increase in relative total immunolabeled nuclei was seen in the CA1 and CA3 regions, but not in the dentate gyrus or hilus of the dentate gyrus. Acute E2 treatment resulted in increased CRE-DNA binding in the frontal cortex but not in amygdala, hippocampus, or cerebellum. However, no changes in levels of total CREB or pCREB protein were observed in the frontal cortex under E2 treatment. No changes were observed either in basal or cAMP-stimulated protein kinase A (PKA) activity or in PKA-α catalytic subunit immunoreactivity in the amygdala or the frontal cortex. Our study indicates that both long-term and acute treatments with estrogens influence the function of CREB in specific brain structures.