Coupling of functional GABA_B receptors (GABA_BR) to G proteins was investigated using the expression system of baby hamster kidney (BHK) cells and Xenopus oocytes. Fluorescent resonance energy transfer (FRET) analysis of BHK cells coexpressing GABA_B1areceptor (GB_1aR) fused to Cerulean, a brighter variant of cyan fluorescent protein, and GABA_B2 receptor (GB₂R) fused to Venus, a brighter variant of yellow fluorescent protein, revealed that GB_1aR-Cerulean and GB₂R-Venus form a heterodimer. The GABA_BR agonists, baclofen and 3-aminopropylphosphinic acid (3-APP), elicited inward rectifying K⁺ currents in a concentration-dependent manner in oocytes expressing GB_1aR and GB₂R, or GB_1aR-Cerulean and GB₂R-Venus, together with G protein activated-inwardly rectifying K⁺ channels (GIRK), but not in oocytes expressing GB_1aR alone or GB₂R alone together with GIRKs. Oocytes coexpressing GB_1aR + G_i2-fused GB₂R (GB₂R-G_i2) caused faster K⁺ currents in response to baclofen. Furthermore, oocytes coexpressing GB_1aR + GB₂R fused to G_qi5 (a chimeric G_q protein that activates phospholipase C [PLC] pathways) caused PLC-mediated Ca²⁺-activated Cl^- currents in response to baclofen. In contrast, these responses to baclofen were not observed in oocytes coexpressing GB_1aR-G_i2 or GB_1aR-G_qi5, together with GB₂R. BHK cells and oocytes coexpressing GB_1aR-Cerulean + triplet tandem of GB₂R-Venus-G_qi5 caused FRET and Ca²⁺-activated Cl^- currents, respectively, with a similar potency in BHK cells coexpressing GB_1aR-Cerulean + GB₂R-Venus and in oocytes coexpressing GB_1aR + GB₂R-G_qi5. Our results indicate that functional GABA_BR forms a heterodimer composed of GB₁R and GB₂R, and that the signal transducing G proteins are directly coupled to GB₂R, but not to GB₁R.