High affinity binding sites (Kd = 1.7 nM) for [3H] imipramine have been characterized in membranes prepared from human brain. The binding of [3H] imipramine was found to be saturable, reversible, and inhibited by pharmacologically active tricyclic antidepressants. Other psychoactive compounds as well as most neurotransmitter substances were ineffective in inhibiting [3H] imipramine binding at concentrations up to 10 μM. The hypothalamus was found to contain a relatively high density of these binding sites and is enriched approximately 4-fold when compared to cerebral and cerebellar cortex. A very good correlation (r = 0.97) p < 0.001 was found between the abilities of a series of clinically active tricyclic antidepressants in displacing specifically bound [3H] imipramine from human brain and platelet membranes, suggesting that the binding sites from these two tissues are very similar.