Regulators of G-protein signaling (RGS) proteins regulate certain G-protein-coupled receptor (GPCR)-mediated signaling pathways. The GABA(B) receptor (GABA(B)R) is a GPCR that plays a role in the stress response. Previous studies indicate that acute immobilization stress (AIS) decreases RGS4 in the prefrontal cortex (PFC) and hypothalamus (HY) and suggest the possibility of a signal complex composed of RGS4 and GABA(B)R. Therefore, in the present study, we tested whether RGS4 associates with GABA(B)R in these brain regions. We found the co-localization of RGS4 and GABA(B)R subtypes in the PFC and HY using double immunohistochemistry and confirmed a direct association between GABA(B2)R and RGS4 proteins using co-immunoprecipitation. Furthermore, we found that AIS decreased the amount of RGS4 bound to GABA(B2)R and the number of double-positive cells. These results indicate that GABA(B)R forms a signal complex with RGS4 and suggests that RGS4 is a regulator of GABA(B)R.