In seven-transmembrane (7TM), G protein-coupled receptors, highly conserved residues function as microswitches, which alternate between different conformations and interaction partners in an extended allosteric interface between the transmembrane segments performing the large scale conformational changes upon receptor activation. Computational analysis using x-ray structures of the β(2)-adrenergic receptor demonstrated that PheVI:09 (6.44), which in the inactive state is locked between the backbone and two hydrophobic residues in transmembrane (TM)-III, upon activation slides ∼2 Å toward TM-V into a tight pocket generated by five hydrophobic residues protruding from TM-III and TM-V. Of these, the residue in position III:16 (3.40) (often an Ile or Val) appears to function as a barrier or gate for the transition between inactive and active conformation. Mutational analysis showed that PheVI:09 is essential for the constitutive and/or agonist-induced signaling of the ghrelin receptor, GPR119, the β(2)-adrenergic receptor, and the neurokinin-1 receptor. Substitution of the residues constituting the hydrophobic pocket between TM-III and TM-V in the ghrelin receptor in four of five positions impaired receptor signaling. In GPR39, representing the 12% of 7TM receptors lacking an aromatic residue at position VI:09, unchanged agonist-induced signaling was observed upon Ala substitution of LeuVI:09 despite reduced cell surface expression of the mutant receptor. It is concluded that PheVI:09 constitutes an aromatic microswitch that stabilizes the active, outward tilted conformation of TM-VI relative to TM-III by sliding into a tight hydrophobic pocket between TM-III and TM-V and that the hydrophobic residue in position III:16 constitutes a gate for this transition.