In gastrointestinal smooth muscle cells, VPAC₂receptor desensitization is exclusively mediated by G protein coupled receptor kinase 2 (GRK2). The present study examined the mechanisms by which acetylcholine (ACh) acting via m3 receptors regulates GRK2-mediated VPAC₂ receptor desensitization in gastric smooth muscle cells. Vasoactive intestinal peptide (VIP) induced VPAC₂ receptor phosphorylation, internalization, and desensitization in both freshly dispersed and cultured smooth muscle cells. Co-stimulation with ACh in the presence of m2 receptor antagonist (i.e., activation of m3 receptors) inhibited VPAC₂ receptor phosphorylation, internalization, and desensitization. Inhibition was blocked by the selective protein kinase C (PKC) inhibitor, bisindolylmaleimide, suggesting that the inhibition was mediated by PKC, derived from m3 receptor activation. Similar results were obtained by direct activation of PKC with phorbol myristate acetate (PMA). In the presence of the m2 receptor antagonist, ACh induced phosphorylation of RKIP (Raf Kinase Inhibitory Protein), increased RKIP:GRK2 association, decreased RKIP:Raf-1 association, and stimulated ERK1/2 activity, suggesting that upon phosphorylation by PKC, RKIP dissociates from its known target Raf to associate with, and block the activity of, GRK2. In muscle cells expressing RKIP(S153A), which lacks the PKC phosphorylation site, RKIP phosphorylation was blocked and the inhibitory effect of ACh on VPAC₂ receptor phosphorylation, internalization and desensitization, as well as the stimulatory effect on ERK1/2 activation were abolished. This study identified a novel mechanism of cross-regulation of G_s-coupled receptor phosphorylation and internalization by G_q-coupled receptors. The mechanism involved phosphorylation of RKIP by PKC, switching RKIP from association with Raf-1 to association with, and inhibition of, GRK2.