Using radioreceptor assay with [3H]ce-bungarotoxin (a-BGT) and [3H]phencyclidine (PCP) as probes for the nicotinic acetylcholine receptor (nAChR) in membranes obtained from honeybee heads, the effects of various nAChR ligands, nicotinoids and neonicotinoids were studied. The data indicated differences in pharmacological characteristics between Torpedo electric organ and honeybee brain nAChRs. [3H]a-BGT binds to the acetylcholine (ACh) recognition site of the nAChRs of vertebrate skeletal muscle, Torpedo electric organ and honeybee brain. In vertebrates, [3H]PCP binds to an allosteric site on the receptors ion channel and its binding is stimulated by receptor activation with agonists. However, the tested vertebrate cholinergic agonists inhibited [3H]a-BGT binding, but did not activate [3H]PCP binding to the honeybee nAChR. Saturation isotherms of the binding of [3H]a-BGT with or without PCP indicated that PCP interacted with the ACh recognition site on the nAChR. Furthermore, nicotine inhibited not only [3H]a-BGT binding but also [3H]PCP binding. Detailed study of [3H]PCP binding indicated that [3H]PCP bound to the honeybee brain membranes both at high and low affinity sites. The former corresponded to the vertebrate allosteric site on the nAChR and the latter to the ACh recognition site. Nicotine, anabasine and nitenpyram bound to both sites, while imidacloprid, 6-Cl-PMNI and acetamiprid bound selectively to the ACh recognition site. In houseflies, nicotine and imidacloprid produced excitation followed by paralysis, while PCP was anesthetic, even though PCP was as insecticidal as nicotine.