Recent studies have demonstrated the presence of high affinity binding sites for [3H] imipramine in membrane preparations derived from rat brain, human platelet, and human brain. Although initial reports concluded that there was no relationship between these binding sites and the reuptake sites for biogenic amines, subsequent studies in our laboratory suggested a close relationship between the high affinity imipramine binding site and the serotonin uptake or transport site (cf. ref. 9). To further establish whether these binding sites are associated with either platelet or neuronal uptake of serotonin, the relative potencies of a series of tricyclic antidepressants in inhibiting [3H] imipramine binding and serotonin uptake were determined under identical assay conditions. A close correlation between inhibition of serotonin uptake and [3H] imipramine binding was observed (r = 0.99, p < 0.001). In addition, electrolytic lesions of the midbrain raphe produced a decrease in [3H] imipramine binding in hypothalamic synaptosomes that paralleled the decrease in [3H] serotonin uptake. Finally incubation of synaptosomal membranes with 2,8-dinitroimipramine, an irreversible inhibitor of [3H] imipramine binding, produced a dose-dependent decrease in serotonin uptake, without altering the uptake of nonrepinephrine or dopamine. Taken together our results strongly suggest that high affinity binding of ] imipramine selectively labels serotonin uptake sites in brain and platelet.