Acetylcholine is the most abundant excitatory neurotransmitter in the insect brain and the most numerous acetylcholine receptors are the nicotinic ones (nAChRs). The genome sequencing of diverse insect species has demonstrated the existence of at least 10 nAChR genes coding for α and β subunits, suggesting the existence in the insect CNS of several subtypes of nAChRs whose molecular composition and pharmacological properties are still unknown. Insect nAChRs have given rise to an abundance of literature about their sensitivity to neonicotinoid insecticides but only limited data are available on the functional role of nAChRs in insect cognitive functions. The data we have collected on honeybees are the only data that shed light on the role of nAChRs in learning and memory processes. The behavioral response of proboscis extension (PER), which appears when the honeybee perceives sugar, was used to quantify learning and memory performances in associative and non‑associative learning procedures. Habituation of the PER, which consists in ceasing to respond to sucrose upon repetitive antennal sucrose stimulation, was facilitated by the injection into the brain of one of the nicotinic antagonists mecamylamine, alpha‑bungarotoxin (α‑BGT) or methyllycaconitine (MLA). Pavlovian associative protocol was used to condition the PER to olfactory or tactile stimulus after single‑ or multiple‑trial training. Localized brain injections of the nicotinic antagonist mecamylamine were performed before or after one‑trial olfactory learning in the mushroom bodies (MB), the integrative structures of the insect brain. The results showed that the calical input structures of the MB are necessary for the acquisition processes and the output α‑lobe regions are involved in retrieval processes. Brain injection of one of the three nicotinic antagonists mecamylamine, α‑BGT and MLA was combined with single‑ and multiple‑trial olfactory and tactile learning and memory performances were evaluated at long‑ or short‑term intervals. Mecamylamine impaired the acquisition of one‑trial learning and the retrieval of information, regardless of the number of trials during training and the learning modality (olfactory or tactile cues used as conditioned stimulus). Memory performance evaluated at long‑term intervals was decreased by injection of α‑BGT and MLA in multiple‑trial olfactory and tactile experiments. We conclude from these results that at least two subtypes of nAChRs exist in the honeybee brain. The α‑BGT‑sensitive nAChRs are necessary for the formation of long‑term memory and the α‑BGT‑insensitive nAChRs are involved in one‑trial acquisition and in retrieval processes. The hypothesis is put forward that multiple‑trial associative learning triggers activation of the α‑BGT‑sensitive nAChRs that, in turn, activate intracellular events leading to LTM formation.