Others and we have characterized several Gβ-dependent effectors in smooth muscle, including GRK2, PLCβ3, and PI 3-kinase , and have identified various signaling targets downstream of PI 3-kinase , including cSrc, integrin-linked kinase, and Rac1-Cdc42/p21-activated kinase/p38 MAP kinase. This study identified a novel mechanism whereby Gβ acting via PI 3-kinase and cSrc exerts an inhibitory influence on Gα_i activity. The G_i2-coupled opioid receptor agonist, DPDPE, activated cSrc, stimulated tyrosine phosphorylation of Gα_i2, and induced Gα_i2:RGS12 association; all three events were blocked by PI 3-kinase (LY294002) and cSrc (PP2) inhibitors and by expression of the carboxyl terminal sequence of GRK2(495-689), a Gβ-scavenging peptide. Inhibition of forskolin-stimulated cAMP and muscle relaxation by DPDPE was augmented by PP2, LY294002 and a selective PI 3 kinase inhibitor, AS605420. Expression of tyrosine-deficient Gα_i2 mutant (Y69F/Y231F/Y321F) or knockdown of RGS12 blocked Gα_i2 phosphorylation and Gα_i2:RGS12 association, and caused greater inhibition of cAMP. Parallel studies using somatostatin, cyclopentyl adenosine, or acetylcholine to activate, respectively, G_i1-coupled sstr3 receptors, and G_i3-coupled adenosine A1 or m2 receptors elicited cSrc activation, Gα_i1 or Gα_i3 phosphorylation, and Gα_i1:RGS12 or Gα_i3:RGS12 association, and inhibition of cAMP. Inhibition of cAMP and muscle relaxation was greatly increased by AS-605240 and by PP2. The results demonstrate that Gβ-dependent tyrosine phosphorylation of Gα_i1/2/3 by cSrc facilitated recruitment of RGS12, a Gα_i-specific RGS protein with a unique phospho-tyrosine-binding (PTB) domain, resulting in rapid deactivation of Gα_i and facilitation of smooth muscle relaxation.