The long isoform of the phosducin-like protein (PhLPl) is widely expressed in the brain and it is thought to influence G-protein signalling by regulating the activity of Gbetagamma dimers. We show that in the mature nervous system, PhLPl exists as both a 38kDa non-glycosylated isoform and as glycosylated isoforms of about 45, 100 and 150kDa. Additionally, neural PhLPl is subject to serine phosphorylation, which augments upon the activation of Mu-opioid receptors (MORs), as does its association with Gbetagamma subunits and 14-3-3 proteins. While the intracerebroventricular (icv) administration of morphine to mice rapidly reduced the association of MORs with G proteins, it increased the serine phosphorylation of these receptors. Moreover, activated Ca2+/calmodulin-dependent protein kinase II (CaMKII) accumulated in the MOR environment and phosphorylated PhLPl was seen to co-precipitate with these opioid receptors. Opioid-induced phosphorylation of PhLPl was impaired by inhibiting the activity of CaMKII and, in these circumstances, the association of PhLPl with Gbetagamma dimers and 14-3-3 proteins was diminished. Furthermore, these events were coupled with the recovery of G protein regulation by the MORs, while there was a decrease in serine phosphorylation of these receptors and morphine antinociceptive tolerance diminished. It seems that CaMKII phosphorylation of PhLPl stabilizes the PhLPl.Gbetagamma complex by promoting its binding to 14-3-3 proteins. When this complex fails to bind to 14-3-3 proteins, the association of PhLPl with Gbetagamma is probably disrupted by GalphaGDP subunits and the MORs recover control on G proteins.