A series of cell lines stably expressing recombinant nicotinic acetylcholine receptors (nAChRs) has been established by transfection of mammalian (rat) and insect (Drosophila) nicotinic subunit cDNAs. By equilibrium radioligand binding, we have examined the influence of individual subunits upon the affinity of two nicotinic agonists (epibatidine and methylcarbamylcholine), an antagonist (the snake neurotoxin, alpha-bungarotoxin) and a recently developed chloronicotinyl insecticide (imidacloprid). Imidacloprid bound with very low affinity to the rat alpha4/beta2 nAChR but did so with high affinity to hybrid nAChRs containing Drosophila alpha subunits co-assembled with rat beta2. Of the subunit combinations examined, imidacloprid showed highest affinity binding to nAChRs containing the recently identified Drosophila alpha subunit, D alpha3, co-assembled with beta2. In contrast, no specific binding of imidacloprid was detected when D alpha3 was co-expressed with the mammalian neuronal beta4 subunit, or with the muscle-type (gamma or delta) subunits. However, despite the absence of imidacloprid binding to D alpha3/beta4, D alpha3/gamma or D alpha3/delta, these subunit combinations all exhibited high affinity binding of other nicotinic radioligands. Epibatidine showed substantially higher affinity binding to subunit combinations containing neuronal (beta2 or beta4) subunits than it did to combinations containing muscle-type (gamma or delta) subunits. In contrast, alpha-bungarotoxin bound with higher affinity to combinations containing muscle-type subunits. Our results demonstrate that both alpha and non-alpha subunits exert a profound influence upon the affinity of nicotinic ligands for recombinant nAChRs.