In vivo brain microdialysis and high-performance liquid chromatography with electrochemical detection were used to study the effect of different selective α 2 -antagonists on hippocampal norepinephrine (NE) release in freely moving awake rat. Systemic administration (0.5 mg/kg i.p.) of either the α 2AD -antagonist BRL 44408 or the α- 2BC -antagonist ARC 239 did not significantly change the basal release of NE. At a higher dose (5 mg/kg i.p.) ARC 239 was still ineffective, whereas BRL 44408 caused a significant increase of the extracellular level of NE. Similar results were obtained from in vitro perfusion experiments. Rat hippocampal slices were loaded with [ 3 H]NE and the electrical stimulation-evoked release of [ 3 H]NE was determined. The α 2 -antagonists were applied in a concentration range of 10 −8 to 10 −6 M. ARC 239 was ineffective, whereas BRL 44408 significantly increased the electrically induced release of [ 3 H]NE. In agreement with the data of microdialysis and perfusion experiments. BRL 44408 displaced [ 3 H]yohimbine from hippocampal and cortical membranes of rat brain with high affinity whereas ARC 239 was less effective. The pK i values of eight different α 2 -adrenergic compounds showed a very good correlation (r = 0.98, slope = 1.11 P < 0.0001) in hippocampus and frontal cortex where the α 2 -adrenoceptors have been characterized as α 2D -subtype. Our data indicate that hippocampal NE release in rat is regulated by α 2D -adrenoceptors, a species variation of the human α 2A -subtype.