Caveolins act as scaffold proteins in multiprotein complexes and have been implicated in signaling by G protein-coupled receptors. Studies using knockout mice suggest that beta(3)-adrenoceptor (AR) signaling is dependent on caveolin-1, however it is not known whether caveolin-1 is associated with the beta(3)-AR or solely with downstream signaling proteins. We have addressed this question by examining the impact of membrane rafts and caveolin-1 on the differential signaling of mouse beta(3a)- and beta(3b)-AR isoforms that diverge at the distal C-terminus. Only the beta(3b)-AR promotes PTX-sensitive cAMP accumulation. When cells expressing the beta(3a)-AR were treated with filipin III to disrupt membrane rafts, or transfected with caveolin-1 siRNA, the cyclic AMP response to the beta(3)-AR agonist CL316243 became PTX sensitive, suggesting Galphai/o coupling. The beta(3a)-AR C-terminus, SP(384)PLNRF(389)DGY(392)EGARPF(398)PT, resembles a caveolin interaction motif. Mutant beta(3a)-ARs (F389A/Y392A/F398A or P384S/F389A) promoted PTX-sensitive cAMP responses, and in situ proximity assays demonstrated an association between caveolin-1 and the wild type beta(3a)-AR but not the mutant receptors. In membrane preparations, the beta(3b)-AR activated Galphao and mediated PTX-sensitive cAMP responses, whereas the beta(3a)-AR did not activate Galphai/o proteins. The endogenous beta(3a)-AR displayed Galphai/o coupling in brown adipocytes from caveolin-1 knockout mice, or in wild type adipocytes treated with filipin III. Our studies indicate that interaction of the beta(3a)-AR with caveolin inhibits coupling to Galphai/o proteins, and suggest that signaling is modulated by a raft-enriched complex containing the beta(3a)-AR, caveolin-1, Galphas and adenylyl cyclase.