Nicotinic acetylcholine receptors (nAChRs) are the main target of neonicotinoid insecticides, which are widely used in crop protection against insect pests. Electrophysiological and molecular approaches have demonstrated the presence of several nAChR subtypes with different affinities for neonicotinoid insecticides. However, the precise mode of action of neonicotinoids on insect nAChRs remains to be elucidated. Radioligand binding studies with [3H]-α-bungarotoxin and [3H]-imidacloprid have proved instructive in understanding ligand binding interactions between insect nAChRs and neonicotinoid insecticides. The precise binding site interactions have been established using membranes from whole body and specific tissues. In this review, we discuss findings concerning the number of nAChR binding sites against neonicotinoid insecticides from radioligand binding studies on native tissues. We summarize the data available in the literature and compare the binding properties of the most commonly used neonicotinoid insecticides in several insect species. Finally, we demonstrate that neonicotinoid-nAChR binding sites are also linked to biological samples used and insect species.