Abstract: Objective In order to understanding the Cholinergic receptors and its subtype, the desensitization of receptors, the effects of cholinergic agonist on intracellular calcium concentration （Ca²⁺i） of isolated VHCs from the guinea pig were investigated. Methods: Vestibular hair cells （VHC） were isolated from guinea pig crista ampullaris by enzymatic and mechanical methods The influence of cholinergic agonist on Ca²⁺ i was examined using laser scanning confocal microscopy（LSCM） and the Ca²⁺ sensitive dye Fluo-3. Results: ①Different concentration of acetylcholine（ACh） and carbachol（CCh）, a muscarnic and nicotinic agonist, induced Ca²⁺i increase with distinctive extent in all VHCs. However, acetycholine Bromide（ACh-Br）, a nicotinic agonist, induced i responses only in some （4/5） of VHCs when 10mmol/L of ACh-Br was applied. No significant Ca²⁺i increase was observed when 1mmol/L of ACh-Br was applied②The desensitization of VHCs was observed when ACh or CCh successively applying to a VHC. In contrast, different type agonist successively applying to VHCs could prevent the desensitization. ③The ACh or CCh induced Ca²⁺ response was suppressed by atropine. In the presence of 0.1mmol/L atropine, the amplitudes of ACh or CCh induced Ca²⁺ response became smaller than that in atropine free medium There is a statistical difference （P＜0.01） Conclusion: The results indicated the presence of cholinergic receptor on guinea pig VHCs. Muscarnic agonist was more responsive for i variation than nicontic receptors. Atropine can suppress muscarnic agonist,induced Ca²⁺ responses. The desensitization of muscarnic receptors to successive stimulations was significant.