Agonist-induced receptor phosphorylation is an initial step in opioid receptor desensitization, a molecular mechanism of opioid tolerance and dependence. Our previous research suggested that agonist-induced delta-opioid receptor (DOR) phosphorylation occurs at the receptor carboxyl terminal domain. The current study was carried out to identify the site of DOR phosphorylation during agonist stimulation and the kinases catalyzing this reaction. Truncation (Delta15) or substitutions (T358A, T361A, and S363G single or triple mutants) at the DOR cytoplasmic tail caused 80 to 100% loss of opioid-stimulated receptor phosphorylation, indicating that T358, T361, and S363 all contribute and are cooperatively involved in agonist-stimulated DOR phosphorylation. Coexpression of GRK2 strongly enhanced agonist-stimulated phosphorylation of the wild-type DOR (WT), but Delta15 or mutant DOR (T358A/T361A/S363G) failed to show any detectable phosphorylation under these conditions. These results demonstrate that T358, T361, and S363 are required for agonist-induced and GRK-mediated receptor phosphorylation. Agonist-induced receptor phosphorylation was severely impaired by substitution of either T358 or S363 with aspartic acid residue, but phosphorylation of the T361D mutant was comparable with that of WT. In the presence of exogenously expressed GRK2, phosphorylation levels of T358D and S363D mutants were approximately half of that of WT, whereas significant phosphorylation of the T358/S363 double-point mutant was not detected. These results indicate that both T358 and S363 residues at the DOR carboxyl terminus are capable of serving cooperatively as phosphate acceptor sites of GRK2 in vivo. Taken together, we have demonstrated that agonist-induced opioid receptor phosphorylation occurs exclusively at two phosphate acceptor sites (T358 and S363) of GRK2 at the DOR carboxyl terminus. These results represent the identification of the GRK phosphorylation site on an opioid receptor for the first time and demonstrate that GRK is the prominent kinase responsible for agonist-induced opioid receptor phosphorylation in vivo.