The distribution of binding sites for the potent inhibitor of norepinephrine (NE) reuptake, [3H]tomoxetine, was examined in rat brain using quantitative autoradiography. Scatchard analysis of [3H]tomoxetine-binding to slide-mounted sections of rat forebrain indicated that the ligand bound to two sites, a high-affinity site with a Kd of 0.29 nM and a lower-affinity site with a Kd of 16 nM. Pharmacological characterization of this high-affinity site was consistent with labelling a NE-uptake site in brain. Autoradiographic localization of the binding sites for [3H]tomoxetine was performed at a ligand concentration of 1 nM representing the distribution of high-affinity sites. The radioligand bound with a distribution of binding sites that was consistent with the known distribution of NE-containing neurons. The highest levels of binding were seen in regions, such as the locus coeruleus, bed nucleus of the stria terminalis, anterior ventral nucleus of the thalamus and the paraventricular nucleus of the hypothalamus. Low levels were seen in regions such as the caudate-putamen, ventral tegmental area and zona reticulata of the substantia nigra, where NE-containing neurons have been reported to be low. Binding to all these sites was inhibited by 1 microM desipramine which produced autoradiograms with a uniform nonspecific binding. These results indicate that low concentrations of [3H]tomoxetine can be used to localize and characterize NE-binding sites. Further study will be necessary to determine the nature of the low-affinity binding site.