Hemicholinium-3 is primarily used in research settings to study the mechanisms of cholinergic neurotransmission. It acts as a competitive inhibitor of the high-affinity choline uptake system, effectively reducing the synthesis of acetylcholine in nerve terminals. This property makes it a valuable tool for investigating the role of acetylcholine in various physiological and pathological processes, such as memory, learning, and neuromuscular function. Additionally, it is utilized in experimental models to explore conditions like myasthenia gravis and other disorders related to cholinergic signaling. Its application helps in understanding the impact of reduced acetylcholine levels on the nervous system and in developing potential therapeutic strategies.