We recently demonstrated that prolonged treatment with morphine increases the antinociceptive potency of the δ-opioid receptor (δOR) agonist deltorphin and promotes cell surface targeting of δORs in neurons of the dorsal horn of the rat spinal cord (Cahill et al., 2001b). In the present study we examined whether these effects were mediated selectively via μOR. Using the same intermittent treatment regimen as for morphine, we found that methadone and etorphine, but not fentanyl, enhanced [ d -Ala 2 ]-deltorphin-mediated antinociception. However, continuous delivery of fentanyl for 48 hr resulted in augmented δOR-mediated antinociception when compared with saline-infused animals. Time course studies confirmed that a 48 hr treatment with morphine was necessary for the establishment of enhanced δOR-mediated antinociception. The observed increases in δOR agonist potency and δOR plasma membrane density were reversed fully 48 hr after discontinuation of morphine injections. Wild-type C57BL/6 mice pretreated with morphine for 48 hr similarly displayed enhanced δOR-mediated antinociception in a tonic pain paradigm. Accordingly, the percentage of plasma membrane-associated δOR in the dorsal horn of the spinal cord, as assessed by immunogold electron microscopy, increased from 6.6% in naive to 12.4% in morphine-treated mice. In contrast, morphine treatment of μOR gene knock-out (KO) mice did not produce any change in δOR plasma membrane density. These results demonstrate that selective activation of μOR is critical for morphine-induced targeting of δOR to neuronal membranes, but not for basal targeting of this receptor to the cell surface.