Effective medical treatment of opiate addiction is limited by a high relapse rate in abstinent addicts. Opiate withdrawal causes cAMP superactivation, but the underlying molecular mechanisms are not clear. Recent evidence implicates an activator of G-protein signaling 3 (AGS3) in opiate addiction. We found previously that during a 10-min activation of opioid receptors, AGS3 binds G alpha(i)-GDP to promote free G betagamma stimulation of adenylyl cyclase (AC) 2 and 4, and/or inactivate G alpha(i) inhibitory function, thereby transiently enhancing cAMP-dependent protein kinase A (PKA) activity. In contrast, we report here that in nucleus accumbens/striatal neurons, morphine withdrawal induces cAMP superactivation, which requires up-regulation of AGS3. cAMP increases as a function of withdrawal time, by approximately 20% at 10 min and 75% at 5 h. However, cAMP superactivation does not require G betagamma. Instead, adenosine A2A receptor activation of G alpha(s/olf) seems to initiate cAMP superactivation and promote AGS3 up-regulation. Elevated AGS3 binds to G alpha(i) to prevent its inhibition on AC activation. Moreover, withdrawal-induced increases in cAMP/PKA activate phospholipase C and epsilon protein kinase C to further stimulate AC5 and AC7, causing cAMP superactivation. Our findings identify a critical role for AC 5 and 7 and A2A receptors for up-regulation of AGS3 in morphine withdrawal-induced cAMP superactivation.