Tricyclic antidepressants (TCAs) have been reported to interact with the opioid system, but their pharmacological activity at opioid receptors has not yet been elucidated. In the present study, we investigated the actions of amoxapine, amitriptyline, nortriptyline, desipramine, and imipramine at distinct cloned and native opioid receptors. In Chinese hamster ovary (CHO) cells expressing ␦-opioid receptors (CHO/DOR), TCAs displaced [3H]naltrindole binding and stimulated guanosine 5Ј-O-(3-[35 S]thio)triphosphate ([ 35 S]GTP␥S) binding at micromolar concentrations with amoxapine displaying the highest potency and efficacy. Amoxapine and amitriptyline inhibited cyclic AMP formation and induced the phosphorylation of signaling molecules along the extracellular signal-regulated kinase 1/2 (ERK1/2) and phosphatidylinositol-3 kinase pathways. Amoxapine also activated ␦-opioid receptors in rat dorsal striatum and nucleus accumbens and human frontal cortex. In CHO cells expressing -opioid receptors (CHO/KOR), TCAs, but not amoxapine, exhibited higher receptor affinity and more potent stimulation of [35 S]GTP␥S binding than in CHO/DOR and effectively inhibited cyclic AMP accumulation. Amitriptyline regulated ERK1/2 phosphorylation and activity in CHO/KOR and C6 glioma cells endogenously expressing -opioid receptors, and this effect was attenuated by the -opioid antagonist nor-binaltorphimine. In rat nucleus accumbens, amitriptyline slightly inhibited adenylyl cyclase activity and counteracted the inhibitory effect of the full agonist trans-(Ϫ) -3,4dichloro-N-methyl-N-[2-(1-pyrrolidinyl)cyclohexyl]benzeneacetamide (U50,488). At the cloned -opioid receptor, TCAs showed low affinity and no significant agonist activity. These results show that TCAs differentially regulate opioid receptors with a preferential agonist activity on either ␦ or subtypes and suggest that this property may contribute to their therapeutic and/or side effects.Although the inhibition of presynaptic reuptake of monoamines is considered to be the primary mechanism of action of tricyclic antidepressants (TCAs), it is well established that these drugs can act on multiple molecular targets by affecting the activity of distinct neurotransmitter receptor systems and ion channels (Baldessarini, 2006). These secondary actions have been generally related to TCAs' adverse side effects, although some of them have been proposed to contribute to the therapeutic activity.An interaction with the opioid system has long been shown to be involved in the analgesic and mood-elevating effects of TCAs. A number of studies have reported that the antinociceptive effects of TCAs are reversed by opioid receptor antagonists (Biegon and Samuel, 1980;Gray et al., 1998;Marchand et al., 2003; Benbouzid et al., 2008a,b) and that TCAs potentiate morphine-induced analgesia both in animals (Hamon et al., 1987) and in humans (Micó et al., 2006). In animal behavioral tests predictive of antidepressant effects in humans, such as the forced swimming and learned helplessness tests, th...