Similar to other G protein-coupled receptors, rapid phosphorylation of the δ-opioid receptor in the presence of agonist has been reported. Hence, agonist-induced desensitization of the δ-opioid receptor has been suggested to be via the receptor phosphorylation, arrestin-mediated pathway. However, due to the highly efficient coupling between the δ-opioid receptor and the adenylyl cyclase, the direct correlation between the rates of receptor phosphorylation and receptor desensitization as measured by the adenylyl cyclase activity could not be established. In the current studies, using an ecdysone-inducible expression system to control the δ-opioid receptor levels in HEK293 cells, we could demonstrate that the rate of deltorphin II-induced receptor desensitization is dependent on the receptor level. Only at receptor concentrations ≤90 fmol/mg of protein were rapid desensitizations (t(1/2) <10 min) observed. Apparently, deltorphin II-induced receptor desensitization involves cellular events in addition to receptor phosphorylation. Mutation of Ser363 in the carboxyl tail of the δ-opioid receptor to Ala completely abolished the deltorphin II-induced receptor phosphorylation but not the desensitization response. Although the magnitude of desensitization was attenuated, the rate of deltorphin II-induced receptor desensitization remained the same in the S363A mutant as compared with wild type. Also, the S363A mutant could internalize in the presence of deltorphin II. Only when the agonist-induced clathrin-coated pit-mediated receptor internalization was blocked by 0.4 M sucrose that the deltorphin II-induced receptor desensitization was abolished in the S363A murant. Similarly, 0.4 M sucrose could partially block the agonist-induced rapid desensitization in HEK293 cells expressing the wild type δ-opioid receptor. Taken together, these data supported the hypothesis that rapid desensitization of the δ-opioid receptor involves both the phosphorylation and the internalization of the receptor.