Previously, we reported that the time course for the rapid phosphorylation rate of μ-opioid receptor expressed in human embryonic kidney (HEK)293 cells did not correlate with the slow receptor desensitization rate induced by [D-Ala2,N-MePhe4,Gly-ol5]-enkephalin (DAMGO). However, others have suggested that receptor phosphorylation is the trigger for μ-opioid receptor desensitization. In this study, we demonstrated the relatively slow rate of receptor desensitization could be attributed partially to the recycling of internalized receptor as determined by fluorescence-activated cell-sorting analysis. However, the blockade of the endocytic and Golgi transport events in HEK293 cells with monensin and brefeldin A did not increase the initial rate of receptor desensitization. But the desensitization rate was increased by reduction of the μ-opioid receptor level with β-furnaltrexamine (βFNA). The reduction of the receptor level with 1 μM βFNA significantly increased the rate of etorphine-induced receptor desensitization. By blocking the ability of receptor to internalize with 0.4 M sucrose, a significant degree of receptor being rapidly desensitized was observed in HEK293 cells pretreated with βFNA. Hence, μ- opioid receptor is being resensitized during chronic agonist treatment. The significance of resensitization of the internalized receptor in affecting receptor desensitization was demonstrated further with human neuroblastoma SHSY5Y cells that expressed a low level of μ-opioid receptor. Although DAMGO could not induce a rapid desensitization in these cells, in the presence of monensin and brefeldin A, DAMGO desensitized the μ-opioid receptor's ability to regulate adenylyl cyclase with a t(1/2) = 9.9 ± 2.1 min and a maximal desensitized level at 70 ± 4.7%. Furthermore, blockade of receptor internalization with 0.4 M sucrose enhanced the DAMGO-induced receptor desensitization, and the inclusion of monensin prevented the resensitization of the μ-opioid receptor after chronic agonist treatment in SHSY5Y cells. Thus, the ability of the μ-opioid receptor to resensitize and to recycle, and the relative efficiency of the receptor to regulate adenylyl cyclase activity, contributed to the observed slow rate of μ-opioid receptor desensitization in HEK293 cells.