Activation of the opioid receptor results in short-term inhibition of intracellular cAMP levels followed by receptor desensitization and subsequent increase of cAMP above the control level (adenylyl cyclase superactivation). Using adenovirus to deliver pertussis toxin-insensitive mutants of the α-subunits of Gi/o that are expressed in neuroblastoma Neuro2A cells (Gαi2, Gαi3, and Gαo), we examined the identities of the G proteins involved in the short- and long-term action of the δ-opioid receptor (DOR). Pertussis toxin pretreatment completely abolished the ability of [D-Pen2,D-Pen 5]-enkephalin (DPDPE) to inhibit forskolin-stimulated intracellular cAMP production. Expression of the C352L mutant of Gαi2, and not the C351L mutants of Gαi3 or Gαo, rescued the short-term effect of DPDPE after pertussis toxin treatment. The ability of Gαi2 in mediating DOR inhibition of adenylyl cyclase activity was also reflected in the ability of Gαi2, not Gαi3 or Gαo, to coimmunoprecipitate with DOR. Coincidently, after long-term DPDPE treatment, pertussis toxin treatment eliminated the antagonist naloxone-induced superactivation of adenylyl cyclase activity. Again, only the C352L mutant of Gαi2 restored the adenylyl cyclase superactivation after pertussis toxin treatment. More importantly, the C352L mutant of Gαi2 remained associated with DOR after long-term agonist and pertussis toxin treatment whereas the wild-type Gαi2 did not. These data suggest that Gαi2 serves as the signaling molecule in both DOR-mediated short- and long-term regulation of adenylyl cyclase activity.