The uptake sites for norepinephrine (NE) in brain have not been studied in much detail, probably due to the absence of an adequate radioligand for labeling these sites. This study describes the binding properties of [3H]nisoxetine to uptake sites for NE in rat brain homogenates and in tissue slices analyzed by quantitative autoradiography. The binding of [3H]nisoxetine was found to be saturable and sodium-dependent to a single class of binding sites (Kd = 0.8 nM). The potencies of drugs to inhibit the uptake of NE correlated highly with their potencies to inhibit the binding of [3H]nisoxetine. Studies using [3H]nisoxetine for mapping of sites associated with uptake of NE by quantitative autoradiography indicated that the pattern of binding of [3H] nisoxetine is consistent with the pattern of noradrenergic innervation. Destruction of central noradrenergic neurons by 6-OH-dopamine or DSP-4 resulted in large decreases in the binding of [3H]nisoxetine in almost all areas of the brain regions examined. [3H]Nisoxetine should prove to be a useful tool to study the regulation of uptake sites for NE as well as a useful marker for noradrenergic innervation in the study of various neurological diseases.