The serine/threonine protein kinase glycogen synthase kinase-3 (GSK-3) has recently been identified as one of the key targets of mood stabilizing agents and antipsychotic drugs. These drugs have been found to promote the phosphorylation at serine 9 (Ser9) and the consequent inactivation of GSK-3β, thereby inhibiting GSK-dependent intracellular signaling. We have previously shown that N-desmethylclozapine (NDMC), a major metabolite of the atypical antipsychotic clozapine, is a potent and efficacious ð opioid receptor agonist. In the present study, we investigated whether NDMC regulates GSK-3β phosphorylation by acting at δ opioid receptor (DOR). We found that in Chinese hamster ovary cells transfected whith the human DOR and in NG108-15 neuroblastoma x glioma hybrid cells expressing endogenous DOR NDMC induced a rapid phosphorylation of GSK-3β at Ser9. This effect was mimicked by the δ opioid receptor ogonist DPDPE and was blocked by naloxone. In addition, in both cell systems NDMC and DPDPE induced the phosphorylation of the protein kinase Akt-1. Pretreatment of cells with the phosphatidylinositol 3 kinase (PI3K) inhibitor worthmannin markedly reduced the stimulatory effect of NDMC on GSK-3β and Akt-1 phosphorylation. These data indicate that NDMC can regulate GSK-dependent signaling by stimulating δ opioid receptors coupled to the PI3K/AKt pathway and suggest that this property may contribute to the antidepressant and atypical antipsychotic activity of clozapine.