By using combined in vitro radioreceptor binding and autoradiographic techniques and [3H]spiroperidol (in the presence of the 5-hydroxytryptamine-2 receptor blocker ketanserin) as a ligand, the pharmacological properties and the anatomical localization of dopamine (DA) DA-2 receptors sites in rat mesenteric vascular tree were analyzed. [3H]Spiroperidol was bound by sections of rat mesentery in a manner consistent with the labeling of DA-2 receptors, with a Kd value of 2.48 nM and with a maximum binding value of 159 fmol/mg of protein. Light microscopic autoradiography revealed specific [3H]spiroperidol binding sites primarily in the adventitial layer and in the adventitial-medial border as well as in the intimal layer. Adventitial and adventitial-medial binding sites disappeared after 6-hydroxydopamine sympathectomy. In contrast, chemical sympathectomy was without effect on intimal [3H]spiroperidol binding sites. The density of adventitial and adventitial-medial binding sites was higher in medium and small sized vessels than in larger ones. These findings are indicative of the existence of DA-2 receptors sites located both prejunctionally (6-hydroxydopamine-sensitive) and in the intimal layer. Prejunctional mesenteric DA-2 receptors may be involved in the inhibition of noradrenaline release from sympathetic neuroeffector junctions. The functional significance of intimal [3H]spiroperidol binding sites, if any, should be established in future studies.