Prolonged opioid exposure occurs frequently as a result of clinical use or drug abuse. Research using different ligands, cell lines, and animal models in the past three decades has elucidated some correlation between the biochemical events and behavioral changes resulting from opioid tolerance, dependence and addiction. For the most part, opioid tolerance and dependence are associated with up-regulation of the cAMP pathway, mediated by the supersensitization of adenylyl cyclase and by the altered coupling of opioid receptors to stimulatory G proteins. Neuroadaptive changes in signal transduction following prolonged opioid exposure are mediated by protein kinase systems, such as protein kinase C (PKC), cyclic AMP-dependent protein kinase (PKA), Ca2+/camodulin-dependent protein kinase II (CaMKII), G protein-coupled receptor kinases (GRKs) and mitogen-activated protein kinases (MAPKs). Intermediate steps between opioid receptor activation and the second- or third-messenger cascades include GRK-mediated receptor endocytosis and intracellular trafficking, as well as interactions with excitatory amino acid receptors and regulation of nitric oxide synthesis. Thus, prolonged occupancy by opioid receptor agonists can have differential effects on opioid receptor internalization, down-regulation and desensitization, and in the supersensitization of adenylyl cyclase, which contribute to the development of opioid tolerance and dependence. We discuss the role of various protein kinases in the signaling mechanisms underlying these differences. Clearer understanding of the molecular mechanisms of opioid tolerance and dependence will help in the treatment of patients suffering from acute and chronic pain, or drug dependence and addiction.