In smooth muscle of the gut, G_q-coupled receptor agonists activate PLC-1 to stimulate phosphoinositide (PI) hydrolysis and IP₃ generation, and induce IP₃-dependent Ca²⁺ release. Inhibition of Ca²⁺ mobilization by PKA and PKG reflects inhibition of PI hydrolysis by both kinases, and PKG-specific inhibitory phosphorylation of IP₃R-I. The mechanism of inhibition of PLC-1-dependent PI hydrolysis has not been established. Neither G_q nor PLC-1 was directly phosphorylated by PKA or PKG in gastric smooth muscle. However, both kinases: (i) phosphorylated RGS4 and induced its translocation from cytosol to plasma membrane, (ii) enhanced acetylcholine (ACh)-stimulated association of RGS4 and G_q.GTP and intrinsic G_q-GTPase activity, and (iii) inhibited ACh-stimulated PI hydrolysis. RGS4 phosphorylation and inhibition of PI hydrolysis were blocked by selective PKA and PKG inhibitors. Expression of RGS4(S52A), which lacks a PKA/PKG phosphorylation site, blocked the increase in GTPase activity and the decrease in PI hydrolysis induced by PKA and PKG. Blockade of PKA-dependent effects was partial. Selective phosphorylation of GRK2, which contains a RGS domain, by PKA augmented ACh-stimulated GRK2:G_q.GTP association; both effects were blocked in cells expressing GRK2(S685A), which lacks a PKA phosphorylation site. Inhibition of PI hydrolysis induced by PKA was partly blocked in cells expressing GRK2(S685A), and completely blocked in cells co-expressing GRK2(S685A) and RGS4(S52A) or G_q(G188S), a G_q mutant that binds GRK2 but not RGS4. The results demonstrate that inhibition of PLC-1-dependent PI hydrolysis by PKA is mediated via stimulatory phosphorylation of RGS4 and GRK2, leading to rapid inactivation of G_q.GTP. PKG acts only via phosphorylation of RGS4.