Opioids produce a number of acute effects, notably antinociception and euphoria, whereas chronic use produces tolerance and dependence. Mu opioid receptors (MOR) mediate opioid antinociception and reinforcement and are distributed throughout the CNS in regions consistent with their acute effects, including striatum, thalamus, amygdala, periaqueductal gray (PAG), locus coeruleus (LC), and spinal cord. G-protein-coupled receptor (GPCR) adaptation involves G-protein-coupled receptor kinase (GRK)-mediated receptor phosphorylation with subsequent β-arrestin binding, which uncouples GPCRs from G-protein activation. β-arrestin binding can initiate receptor endocytosis, leading to subsequent degradation or recycling of GPCRs. These processes are reflected experimentally by desensitization (loss of functional response) and downregulation (loss of receptor binding sites). Evaluation of MOR levels following chronic opioid treatment has indicated that MOR downregulation is not required for the expression of tolerance and dependence. In contrast, evaluation of post-receptor events has revealed region-dependent alterations in MOR-G-protein coupling and subsequent effector activity. For example, MOR-mediated G-protein activity and inhibition of adenylyl cyclase are generally decreased in brainstem nuclei following chronic morphine administration, whereas no changes are found in striatum. Similarly, tolerance develops to MOR-mediated potassium channel activation in regions that include LC and PAG. Chronic opioid-mediated effects on adenylyl cyclase affect downstream signaling via regulation of cAMP dependent protein kinase (PKA) and cAMP response element binding protein (CREB), both of which are altered in LC, nucleus accumbens (NAC) and amygdala, regions that might contribute to motivational and physiological signs of withdrawal. Possible explanations for regional differences in MOR adaptation include region-specific co-localization of MOR with signaling proteins and differential distribution of MOR splice variants or oligomers. Moreover, the finding that regional differences exist in neuroadaptation suggests that behavioral adaptations to chronic opioid administration will vary based on the functional neuroanatomy of the MOR system.