Antidepressant mechanisms of action remain shrouded in mystery, greatly hindering our ability to develop therapeutics which can fully treat patients, suffering from depressive disorders. In an attempt to shed new light on this topic, we have undertaken a series of studies investigating actions of tricyclic antidepressant drugs (TCAs) at the alpha(2A) adrenergic receptor (AR), a centrally important receptor, dysregulation of which has been linked to depression. Our previous work established a particular TCA, desipramine, as an arrestin-biased alpha(2A)AR ligand driving receptor endocytosis and downregulation but not canonical heterotrimeric G protein-mediated signaling. The present work is aimed at broadening our understanding of how members of the TCA drug class act at the alpha(2A)AR, as we have selected the closely related but subtly different TCAs imipramine and amitriptyline for evaluation. Our data demonstrate that these drugs do also function as direct arrestin-biased alpha(2A)AR ligands. However, these data reveal differences in receptor affinity and in the extent/nature of arrestin recruitment to and endocytosis of alpha(2A)ARs. Specifically, amitriptyline exhibits an approximately 14-fold stronger interaction with the receptor, is a weaker driver of arrestin recruitment, and preferentially recruits a different arrestin subtype. Extent of endocytosis is similar for all TCAs studied so far, and occurs in an arrestin-dependent manner, although imipramine uniquely retains a slight ability to drive alpha(2A)AR endocytosis in arrestin-null cells. These findings signify an important expansion of our mechanistic understanding of antidepressant pharmacology, and provide useful insights for future medicinal chemistry efforts. (C) 2014 Elsevier Inc. All rights reserved.