Positive allosteric modulators (PAMs) of α4β2 nicotinic acetylcholine receptors have the potential to improve cognitive function and alleviate pain, but the mechanisms of modulation are not well understood. We have investigated two structurally diverse PAMs at α4β2 receptors using two-electrode voltage-clamp electrophysiology and molecular biology. We show that the two PAMs produce distinct pharmacological fingerprints which for NS206 is amplification of current amplitudes above the level of maximal ACh stimulations (Emax) and with no or limited effects at potencies and current wave-forms. The other PAM, NS9283, has a benzodiazepine like profile and acts by left-shifting the ACh concentration response relationship (CRR) without altering Emax. While NS206 was observed to positively modulate ACh-evoked currents at both known α4β2 stoichiometries (2α:3β and 3α:2β), NS9283 only modulates the 3α:2β receptor and is therefore stoichiometry selective. Interestingly, the two modulators can act simultaneously in an additive manner at 3α:2β receptors which results in current levels exceeding Emax of ACh alone and a left-shifted ACh CRR, a combination of the characteristics of each modulators. Through introduction of point mutants and using chimeric receptors, the binding site of NS206 was linked to the interface between the α4-subunit extracellular and transmembrane domains while binding of NS9283 was shown to be associated with the αα-interface in 3α:2β receptors. Collectively, these data demonstrate the existence of two distinct modulatory sites in α4β2 receptors with unique pharmacological properties that can act additively. The two sites appear to be conserved among many members of the Cys-loop receptor family. A detailed picture of allosteric modulatory mechanisms and binding sites of these important receptors is emerging and opens up new avenues for rational drug discovery.