Morphine is a powerful pain reliever, but also a potent inducer of tolerance and dependence. The development of opiate tolerance occurs on continued use of the drug such that the amount of drug required to elicit pain relief must be increased to compensate for diminished responsiveness. In many systems, decreased responsiveness to agonists has been correlated with the desensitization of G-protein-coupled receptors. In vitro evidence indicates that this process involves phosphorylation of G-protein-coupled receptors and subsequent binding of regulatory proteins called β-arrestins. Using a knockout mouse lacking β-arrestin-2 (βarr2-/-), we have assessed the contribution of desensitization of the μ-opioid receptor to the development of morphine antinociceptive tolerance and the subsequent onset of physical dependence. Here we show that in mice lacking β-arrestin-2, desensitization of the μ-opioid receptor does not occur after chronic morphine treatment, and that these animals fail to develop antinociceptive tolerance. However, the deletion of β-arrestin-2 does not prevent the chronic morphine-induced upregulation of adenylyl cyclase activity, a cellular marker of dependence, and the mutant mice still become physically dependent on the drug.