The distribution and physiological functions of the α6 subunit-containing (α6⁎) nicotinic acetylcholine receptors in the central nervous system make them interesting putative therapeutic targets in several disorders. However, investigations into the receptors have been complicated by their inefficient functional expression in vitro. In the present study we have characterized and compared the pharmacological properties displayed by α6β4 and α6β4β3 nicotinic acetylcholine receptors assembled in tsA201 cells from the classical α6/α3 chimera (C1) and two novel α6/α3 chimeras (C6F223L and C16F223L) identified in a recent study ( Jensen et al., 2013). Whereas the Bmax values exhibited by [3H]epibatidine at wild-type α6β4, C1β4, C6F223Lβ4 and C16F223Lβ4 receptors differed substantially, the radioligand and seven orthosteric nicotinic agonists exhibited very similar KD and Ki values at the four receptors. In the FLIPR™ Membrane Potential Blue assay, the agonists exhibited the same rank order of potencies [(±)-epibatidine>sazetidine A>varenicline>(−)-cytisine~(S)-nicotine>acetylcholine>carbachol] at the C1β4, C1β4β3, C6F223Lβ4, C6F223Lβ4β3, C16F223Lβ4 and C16F223Lβ4β3 receptors, albeit the absolute EC50 values differed somewhat between the receptors. Furthermore, four reference antagonists displayed the same rank order of inhibitory potencies at the six receptors [α-conotoxin PIA>2,2,6,6-tetramethylpiperidin-4-yl heptanoate>mecamylamine>dihydro-β-erythroidine]. Although all interpretations based on these results should be made keeping the molecular modifications in the α6 surrogate subunits in mind, this study sheds light on the pharmacological properties of α6β4⁎ nicotinic acetylcholine receptors and demonstrates the applicability of the C6F223L and C16F223L chimeras for studies of these receptors.