We have used the recent tritium-sensitive film method of quantitative autoradiography to localize in rat brain high-affinity binding sites for nitroimipramine (NI), a long-lasting inhibitor of serotonin (5-HT) transport in platelets. The distribution of NI binding sites in rat brain closely parallels the location of 5-HT terminals. There are high concentrations of binding sites in the dorsal and medial raphe nuclei, the basal portion of the mammilary nuclei, the medial forebrain bundle, the olfactory tubercule and the posterior basal nucleus of the amygdala. The association of [3H]NI binding sites with regions having a high content of 5-HT supports the notion that the high-affinity binding site for [3H]NI corresponds to some aspect of the presynaptic uptake site for 5-HT.