Mammalian nicotinic acetylcholine receptors (nAChRs) have been implicated in several neurological disorders and are increasingly being seen as important target sites for therapeutic drug discovery. The identification of subtype-selective nicotinic ligands (including agonists, antagonists and potentiators) is currently a major research goal, both for pharmaceutical drug development and as tools for the pharmacological characterisation of neuronal nAChRs. We have used a variety of molecular pharmacological and electrophysiological techniques to examine the nAChR subtype-selectivity of novel and recently identified nicotinic agonists and potentiators. Heterologous expression of chimeric and mutated subunits is being used to identify the influence of subunit domains and of individual amino acids upon the potency and selectivity of nicotinic ligands. TMAQ (5-(Trifluoromethyl)-6-(1-methyl-azepan-4-yl)methyl-1H-quinolin-2-one) is a β4 subunit-selective nicotinic agonist which also exhibits remarkable species selectivity. Studies with recombinant nAChRs expressed in mammalian cell lines and in Xenopus oocytes have revealed that TMAQ is a potent agonist of nAChRs containing the human β4 subunit but exhibits little or no agonist potency on recombinant nAChRs containing the rat β4 subunit. In contrast, other nicotinic receptor agonists such as epibatidine show little or no difference in their affinity or agonist potency for human and rat β4-contaning nAChRs. With the aim of identifying subunit domains and individual amino acid contributing to the species-selectivity of TMAQ, a series of human-rat β4 subunit chimeras were constructed. In addition, individual amino acids within the human and rat β4 subunits were altered by means of site-directed mutagenesis. Recombinant receptors containing wild-type, chimeric or mutated β4 subunits were examined by radioligand binding, intracellular calcium assays (using a fluorometric imaging plate reader) and by electrophysiological recording. Two adjacent amino acids located close to loop-D in the extracellular domain of the β4 subunit (N57 and V58 in human β4 subunit and S57 and I58 in rat β4) have been identified as playing a critical role in the binding and agonist potency of TMAQ. These findings provide further evidence that non-α subunits can have a substantial influence upon the pharmacological properties of neuronal nAChRs. More recent studies have been aimed at examining the mechanism of action and subtype selectivity of potent nAChR potentiators.