These studies determined whether diabetes and estradiol treatment altered norepinephrine (NE) release from hypothalamus, preoptic area (POA), and cortical slices from ovariectomized (OVX) female rats. Animals were sacrificed 12 days after the onset of streptozotocin-induced diabetes and 48 h following vehicle or estradiol injection. Brain slices were preloaded with <sup>3</sup>H-NE, and release was evoked twice (S1 and S2) by electrical stimulation. Diabetes increased hypothalamic NE release during S1 regardless of the administration of vehicle or estradiol. Neither estradiol treatment nor diabetes alone affected NE release during S2 in the hypothalamus or POA. Estradiol treatment elevated NE release in the POA during S2 but only in diabetic animals. Moreover, estradiol elevated cortical NE release during S2 regardless of the presence or absence of disease. We also examined whether α<sub>2</sub>-adrenoceptor regulation of NE release was influenced by diabetes or hormone treatment. Enhancement of NE release by α<sub>2</sub>-adrenoceptor antagonism was evident in all 3 brain regions. However, α<sub>2</sub>-adrenoceptor regulation of NE release was unaffected by diabetes and hormone treatment. These findings suggest that diabetes alters NE release in the hypothalamus/POA of female rats. Additionally, this work identifies a novel action of estradiol to enhance stimulated NE release in the cortex of female rats.