Alternative splicing of mouse beta3-adrenoceptor transcripts produces an additional receptor isoform (beta3b-adrenoceptor) with a C terminus comprising 17 amino acids distinct from the 13 in the known receptor (beta3a-adrenoceptor). We have shown that the beta3b-adrenoceptor couples to both Gs and Gi, whereas the beta3a-adrenoceptor couples only to Gs. To define the regions involved in this differential G protein coupling, we have compared wild-type, truncated, and mutant beta3-adrenoceptors. In Chinese hamster ovary cells expressing beta3-adrenoceptors truncated at the splicing point, cAMP accumulation with CL316243 [(R,R)-5-[2-[[2-(3-chlorophenyl)-2-hydroxyethyl]-amino]-propyl]1,3-benzodioxole-2,2-dicarboxylate] increased by 59% following pretreatment with pertussis toxin, suggesting that the C-terminal region of the beta3a-adrenoceptor inhibits coupling to Gi. We next utilized the cell-penetrating peptide Transportan 10 (Tp10) to introduce peptides comprising the different C-terminal tail fragments into cells expressing beta3a-adrenoceptor, beta3b-adrenoceptor, and the truncated beta3-adrenoceptor. Treatment with beta3a-Tp10 (1 microM) caused cAMP responses to CL316243 in the beta3a-adrenoceptor to become pertussis toxin-sensitive and display a 30% increase over control, whereas the other peptides did not affect any receptor. Mutation at a potential tyrosine phosphorylation site (Tyr392Ala beta3a-adrenoceptor) did not alter responses or pertussis toxin sensitivity relative to the parent receptor. Surprisingly, a Ser388Ala/Ser389Ala mutant beta3b-adrenoceptor became unresponsive to CL316243 while retaining an extracellular acidification rate response to SR59230A [3-(2-ethylphenoxy)-1-[(1,S)-1,2,3,4-tetrahydronapth-1-ylamino]-2S-2-propanol oxalate]. Our findings suggest that the beta3a-adrenoceptor cannot couple to Gi because of conformational changes induced by a protein(s) that interacts with residues in the C-terminal tail or because this protein(s) affects the intracellular localization of the beta3a-adrenoceptor.