Optimum assay conditions for the association of [3H]-para-tyramine [( 3H]-pTA) with rat brain membranes were characterized, and a saturable, reversible, drug-specific, and high affinity binding mechanism for this trace amine was revealed. The binding capacity (Bmax) for [3H]-pTA in the corpus striatum was approximately 30 times higher than that in the cerebellum, with similar dissociation constants (KD). The binding process of [3H]-pTA involved the dopamine system, inasmuch as (a) highest binding capacity was associated with dopamine-rich regions; (b) dopamine and pTA equally displaced specifically bound [3H]-pTA; (c) there was a severe loss in striatal binding capacity for [3H]-pTA and, reportedly, for [3H]-dopamine, following unilateral nigrostriatal lesion; (d) acute in vivo reserpine treatment markedly decreased the density of [3H]-pTA and, reportedly, of [3H]-dopamine binding sites. In competition experiments [3H]-pTA binding sites, though displaying nanomolar affinity for dopamine, revealed micromolar affinities for the dopamine agonists apomorphine and pergolide, and for several dopamine antagonists, while having very high affinity for reserpine, a marker for the catecholamine transporter in synaptic vesicles. The binding process of [3H]-pTA was both energy-dependent (ouabain-sensitive), and ATP-Mg2+-insensitive; furthermore, the potencies of various drugs in competing for [3H]-pTA binding to rat striatal membranes correlated well (r = 0.96) with their reported potencies in inhibiting [3H]-dopamine uptake into striatal synaptosomes. It is concluded that [3H]-pTA binds at a site located on/within synaptic vesicles where it is involved in the transport mechanism of dopamine.