Many antidepressant drugs, including the tricyclic antidepressant desipramine (DMI), are broadly understood to function by modulating central noradrenergic neurotransmission. α2 adrenergic receptors (α2ARs) are key regulators of the noradrenergic system, and previous work has implicated α2ARs in mediating the antidepressant activity of DMI in the rodent forced swim test (FST). However, little is known about intracellular regulators of antidepressant drug action. α2AR function is tightly regulated by its intracellular interacting partners arrestin and the dendritic protein spinophilin. We have previously established the competitive and reciprocal nature of these interacting proteins at the α2AR in the context of classic agonist effects, and have shown DMI to be a direct arrestin-biased ligand at the receptor. In the present study, we report that mice deficient in the α2AAR subtype lack DMI-induced antidepressant behavioral effects in the FST. As well, mice deficient in arrestin3 lack antidepressant response to DMI, while spinophilin-null mice have enhanced antidepressant response to DMI compared with wild-type controls, indicating that this α2AAR-mediated response is reciprocally regulated by arrestin and spinophilin. The characteristic of α2AAR-dependence and arrestin3 involvement was shared by the antidepressant effect of the classic α2AR agonist clonidine but not the non-tricyclic norepinephrine reuptake inhibitor reboxetine, supporting a model whereby DMI exerts its antidepressant effect through direct engagement of the α2AAR and arrestin3. Our results implicate arrestin- and spinophilin-mediated regulation of the α2AAR in the pharmacology of the noradrenergic antidepressant DMI, and suggest that manipulation of this mode of receptor regulation may represent a novel and viable therapeutic strategy. © 2011 Elsevier Ltd. All rights reserved.