Alternative splicing of the mouse β3-adrenoceptor (AR) produces two isoforms: the β3a-AR couples to Gs and the β3b-AR couples to both Gs and Gi (Hutchinson et al, 2002). To define the residues involved in this differential G protein coupling, truncated receptors were expressed in CHO-K1 cells and their properties examined. In these cells, maximal cAMP accumulation following stimulation with CL316243 was increased relative to control (control, 1.00±0.07; PTX, 2.20±0.22, n=5, P<0.0001) after pretreatment with pertussis toxin (PTX, 100ng ml-1). These results suggested that the β3a-AR is restrained from coupling to Gi by residues in the C-terminus. This view was supported by studies using a cell penetrating peptide transportan-10 (Tp10) to introduce peptides comprising the β3-AR C-terminal tail fragments into cells. Treatment with β3a-Tp10 caused the β3a-AR to become PTX sensitive, whilst the other peptides did not affect cAMP responses to stimulation of any receptor (Sato et al., 2005). Site directed mutagenesis was used to identify the residues in the C-terminus of the β3a-AR responsible for inhibition of coupling to Gi. Mutation of a putative caveolin binding site caused β3a-AR mediated cAMP accumulation to become PTX sensitive (control, 1.00±0.16; PTX, 1.75±0.14, n=4, P<0.0001). Furthermore, filipin III, an agent that disrupts lipid rafts such as caveolae, also caused the wild type β3a-AR to become PTX sensitive (control, 1.00±0.19; PTX, 1.56±0.04, n=4, P<0.0001), but in addition concentration-response curves to CL316243 were markedly shifted to the right (pEC50 control: 9.76±0.03, +filipin III: 8.73±0.21). To determine if this signalling mechanism had physiological significance we examined the effects of filipin III on mouse brown adipocytes that predominantly express β3a-AR (Chernogubova et al., 2005). CL316243 caused a concentration-dependent increase in cyclic AMP levels in primary brown adipocytes from FVB mice, that was unaffected by pre-treatment of PTX. Filipin III (1μg ml-1) significantly reduced the cAMP response to CL316243 (P<0.0001), indicating that disruption of caveolae limited the Gs/AC/cAMP pathway. Addition of PTX after filipin III treatment caused a significant increase of in the maximal cAMP response to CL316243 compared to control (P<0.05). The study suggests that β3a-ARs are restricted to caveolae and that localization of the receptor may play a specific role in G-protein mediated signalling.