In the hippocampus, signaling through G protein-coupled receptors is modulated by Regulators of G protein signaling (Rgs) proteins, which act to stimulate the rate of GTP hydrolysis, and consequently, G protein inactivation. The R7-Rgs subfamily selectively deactivates the Gi/o-class of Gα subunits that mediate the action of several GPCRs. Here, we used co-immunoprecipitation, electrophysiology and immunoelectron microscopy techniques to investigate the formation of macromolecular complexes and spatial relationship of Rgs7/Gβ5 complexes and its prototypical signaling partners, the GABAB receptor and Girk channel. Co-expression of recombinant GABAB receptors and Girk channels in combination with co-immunoprecipitation experiments established that the Rgs7/Gβ5 forms complexes with GABAB receptors or Girk channels. Using electrophysiological experiments, we found that GABAB-Girk current deactivation kinetics was markedly faster in cells coexpressing Rgs7/Gβ5. At the electron microscopic level, immunolabeling for Rgs7 and Gβ5 proteins was found primarily in the dendritic layers of the hippocampus and showed similar distribution patterns. Immunoreactivity was mostly localized along the extrasynaptic plasma membrane of dendritic shafts and spines of pyramidal cells and, to a lesser extent, to that of presynaptic terminals. Quantitative analysis of immunogold particles for Rgs7 and Gβ5 revealed an enrichment of the two proteins around excitatory synapses on dendritic spines, virtually identical to that of Girk2 and GABAB1. These data support the existence of macromolecular complexes composed of GABAB receptor-G protein-Rgs7-Girk channels in which Rgs7 and Gβ5 proteins may preferentialy modulate GABAB receptor signaling through the deactivation of Girk channels on dendritic spines. In contrast, Rgs7 and Girk2 were associated but mainly segregated from GABAB1 in dendritic shafts, where Rgs7/Gβ5 signaling complexes might modulate Girk-dependent signaling via a different metabotropic receptor(s). © 2013 Wiley Periodicals, Inc.