In neurons, the C terminus of the Mu-opioid receptor (MOR) binds to the protein kinase C-interacting protein/histidine triad nucleotide binding protein 1 (PKCI/HINT1) which in turn binds the regulator of G-protein signalling RGSZ1/Z2 (RGSZ) protein. In this study, we found that intracerebroventricular (icv) administration of morphine recruits PKC isoforms, mostly PKCgamma, to the MOR via the HINT1/RGSZ complex. There, diacylglycerol (DAG) activates this PKCgamma to phosphorylate the MOR and thus, its signal strength was reduced. When PKCI/HINT1 expression is depressed, morphine produces stronger analgesic effects and neither the PKCgamma-MOR complex nor serine phosphorylation of this receptor is detected. This MOR-PKC association involves the cysteine rich domains (CRDs) in the regulatory C1 region of PKC, as well as requiring free zinc ions, HINT1 and RGSZ proteins. Increasing the availability of this metal ion recruits inactive PKCgamma to the MOR, while phorbol esters prevent this binding and even disrupt it. The nitric oxide donor (S)-Nitroso-N-acetylpenicillamine (SNAP) foments the association of PKCgamma with the MORs, effect that was prevented by the heavy metal chelator N,N,N',N'-tetrakis(2-pyridylmethyl) ethylenediamine (TPEN), suggesting a role for endogenous zinc and neural nitric oxide synthase. The N-methyl-D-aspartate receptor (NMDAR) antagonist, MK801, also prevented PKCgamma recruitment to MORs and serine phosphorylation of the receptors following icv morphine. These results indicate that the NMDAR/nNOS cascade, activated via MORs, provide the free zinc ions required for inactive PKCgamma to bind to HINT1/RGSZ complex at the C terminus of the receptor.