The artificial sweetener cyclamate tastes sweet to humans, but not to mice. When expressed in vitro, the human sweet receptor (a heterodimer of two taste receptor subunits: hT1R2 + hT1R3) responds to cyclamate, but the mouse receptor (mT1R2 + mT1R3) does not. Using mixed-species pairings of human and mouse sweet receptor subunits, we determined that responsiveness to cyclamate requires the human form of T1R3. Using chimeras, we determined that it is the transmembrane domain of hT1R3 that is required for the sweet receptor to respond to cyclamate. Using directed mutagenesis, we identified several amino acid residues within the transmembrane domain of T1R3 that determine differential responsiveness to cyclamate of the human versus mouse sweet receptors. Alanine-scanning mutagenesis of residues predicted to line a transmembrane domain binding pocket in hT1R3 identified six residues specifically involved in responsiveness to cyclamate. Using molecular modeling, we docked cyclamate within the transmembrane domain of T1R3. Our model predicts substantial overlap in the hT1R3 binding pockets for the agonist cyclamate and the inverse agonist lactisole. The transmembrane domain of T1R3 is likely to play a critical role in the interconversion of the sweet receptor from the ground state to the active state.