G protein-coupled receptors (GPCRs) selectively couple to specific heterotrimeric G proteins comprised of four subfamilies in order to induce appropriate physiological responses. However, structural determinants in Gα subunits responsible for selective recognition by approximately 800 human GPCRs have remained elusive. Here, we directly compare the influence of subtype-specific Gα structures on the stability of GPCR-G protein complexes and the activation by two Gq-coupled receptors. We used FRET-assays designed to distinguish multiple Go and Gq-based Gα chimeras in their ability to be selectively bound and activated by muscarinic M3 and histaminic H1 receptors. We identify the N-terminus including the αN/β1-hinge, the β2/β3-loop and the α5 helix of Gα to be key selectivity determinants which differ in their impact on selective binding to GPCRs and subsequent activation depending on the specific receptor. Altogether, these findings provide new insights into the molecular basis of G protein-coupling selectivity even beyond the Gα C-terminus.