We showed previously that Gβγ interacts with Receptor for Activated CKinase 1 (RACK1), a protein that not only binds activated protein kinase C (PKC) but also serves as an adaptor/scaffold for many signaling pathways. Here we report that RACK1 does not interact with Gα subunits or heterotrimeric G proteins but binds free Gβγ subunits released from activated heterotrimeric G proteins following the activation of their cognate receptors in vivo. The association with Gβγ promotes the translocation of RACK1 from the cytosol to the membrane. Moreover, binding of RACK1 to Gβγ results in inhibition of Gβγ-mediated activation of phospholipase C β2 and adenylyl cyclase II. However, RACK1 has no effect on other functions of Gβγ, such as activation of the mitogen-activated protein kinase signaling pathway or chemotaxis of HEK293 cells via the chemokine receptor CXCR2. Similarly, RACK1 does not affect signal transduction through the Gα subunits of Gi, Gs, or Gq. Collectively, these findings suggest a role of RACK1 in regulating specific functions of Gβγ.