Heterotrimeric G proteins have been thought to function on the plasma membrane after activation by transmembrane receptors. Here we show that, after activation by receptors, the G protein ␥ complex selectively translocates to the Golgi. Receptor inactivation results in G␥ translocating back to the plasma membrane. Both translocation processes occur rapidly within seconds. The efficiency of translocation is influenced by the type of ␥ subunit present in the G protein. Distinctly different receptor types are capable of inducing the translocation. Receptor-mediated translocation of G␥ can spatially segregate G protein signaling activity.Heterotrimeric (␣␥) G proteins are localized to the plasma membrane of mammalian cells, facilitating interaction and activation by transmembrane G protein-coupled receptors (1-3). Extensive characterization of the effectors on which the G proteins act has suggested that the activated G protein ␣ and ␥ subunits function on the plasma membrane (3-6). It has been thought that the post-translational addition of a lipid moiety to the ␣ subunit and the ␥ subunit aids in the localization of G␣ and G␥ complex to the plasma membrane, where they act on the effector molecules (7). However, there is little information about the properties of these proteins or the signaling they mediate in intact live mammalian cells, because studies attempting to observe G protein function in living mammalian cells have been limited.To visualize the impact of receptor activation and inactivation on the spatial distribution of G protein subunits, we tagged G protein subunits with the yellow and cyan mutant forms of the green fluorescent protein, YFP 1 and CFP, respectively. The fusion proteins were expressed in mammalian cell lines and observed after activating overexpressed or endogenous receptors using fluorescence-based imaging methods. Although we have previously obtained evidence indicating the direct involvement of the G protein ␥ subunit in receptor interaction (8, 9), we examined the effect of receptor activation on ␣o-CFP, 1, and different ␥ subunit types tagged with YFP in Chinese hamster ovary (CHO) cells overexpressing M2 muscarinic receptors. We discovered that ␥-YFP translocated from the plasma membrane to the cell interior on receptor activation and translocated back to the plasma membrane on inactivation. The  subunit co-translocated with the ␥ subunit. The rapidity of the translocation process and proportion of ␥ complex that translocated were dependent on the ␥ subunit type present in the expressed G protein. Experiments using a marker for the Golgi complex and a Golgi disrupting agent, brefeldin A, indicated that the ␥ complex translocates to the Golgi complex. The translocation was sensitive to G i/o -and G q -coupled receptor stimulation. Endogenous receptors also stimulated G␥ translocation. The translocation of the ␥ complex is selective, because ␣o-CFP or a chimeric ␣o-q-CFP that couples to G q -coupling receptors do not translocate from the plasma membrane in response to rece...