Deciphering which specific agonist-receptor interactions affect efficacy levels is of high importance, because this will ultimately aid in designing selective drugs. The novel compound NS3861 and cytisine are agonists of nicotinic acetylcholine receptors (nAChRs) and both bind with high affinity to heteromeric a3ß4 and a4ß2 nAChRs. However, initial data revealed that the activation patterns of the two compounds show very distinct maximal efficacy readouts at various heteromeric nAChRs. To investigate the molecular determinants behind these observations, we performed in-depth patch clamp electrophysiological measurements of efficacy levels at heteromeric combinations of a3- and a4-, with ß2- and ß4-subunits, and various chimeric constructs thereof. Compared with cytisine, which selectively activates receptors containing ß4- but not ß2-subunits, NS3861 displays the opposite ß-subunit preference and a complete lack of activation at a4-containing receptors. The maximal efficacy of NS3861 appeared solely dependent on the nature of the ligand-binding domain, whereas efficacy of cytisine was additionally affected by the nature of the ß-subunit transmembrane domain. Molecular docking to nAChR subtype homology models suggests agonist specific interactions to two different residues on the complementary subunits as responsible for the ß-subunit preference of both compounds. Furthermore, a principal subunit serine to threonine substitution may explain the lack of NS3861 activation at a4-containing receptors. In conclusion, our results are consistent with a hypothesis where agonist interactions with the principal subunit (a) primarily determine binding affinity, whereas interactions with key amino acids at the complementary subunit (ß) affect agonist efficacy.