G protein–coupled receptors (GPCRs) constitute a large and functionally diverse family of transmembrane proteins. They are fundamental in the transfer of extracellular stimuli to intracellular signaling pathways and are among the most targeted proteins in drug discovery. The detailed molecular mechanism for agonist-induced activation of rhodopsin-like GPCRs has not yet been described. Using a combination of site-directed mutagenesis and molecular modeling, we characterized important steps in the activation of the human histamine H1 receptor. Both Ser3.36 and Asn7.45 are important links between histamine binding and previously proposed conformational changes in helices 6 and 7. Ser3.36 acts as a rotamer toggle switch that, upon agonist binding, initiates the activation of the receptor through Asn7.45. The proposed transduction involves specific residues that are conserved among rhodopsin-like GPCRs. © 2005, Nature Publishing Group. All rights reserved.