A number of amino acids essential for Gs coupling, i.e. hot spots, were identified following in vitro Ala-scanning mutagenesis of the cytosolic extensions of helices 3, 5, and 6 and of intracellular loops 2 and 3 of the human luteinizing hormone receptor (LHR). Consistent with the results of in vitro experiments involving ligand binding and ligand-mediated signaling in transiently transfected human embryonic kidney 293 cells, computational modeling of the isolated receptor and of the receptor-G protein complexes suggests an important role of the cytosolic extension of helix 3 and the N-terminal portion of the intracellular loop 2 in Gs interaction, while the contribution of intracellular loop 3 is marginal. Mapping the hot spots into the computational models of LHR and the LHR-Gs complexes allowed for a distinction between receptor sites required for intramolecular structural changes (i.e. I460, T461, H466, and I549) and receptor sites more likely involved in G protein recognition (i.e. R464, T467, I468, Y470, Y550, and D564). The latter sites include the highly conserved arginine of the (E/D)R(Y/W) motif that is therefore likely to be a receptor recognition point for Gs rather than a switch of receptor activation. The results of in vitro and in silico experiments carried out in this study represent the first comprehensive delineation of functionality of the individual residues in the intracellular domains of LHR and establish potential switches of receptor activation as well as a map of the primary receptor recognition sites for Gs. A novel way to consider constitutively active mutants was inferred from this study, i.e. receptor states with improved complementarity for the G protein compared to the wild type receptor.