Molecular biological studies have demonstrated that both muscarinic receptor subtypes and nicotinic receptor subunits were located in mammalian vestibular sensorineural epithelium. However, the functional roles are still unclear, with the exception of the well-known alpha9-containing nicotinic ACh receptor (alpha9nAChR). In this study, the properties of acetylcholine (ACh)-induced currents were investigated by whole-cell patch clamp technique in isolated type II vestibular hair cells (VHCs II) of guinea pig. VHCs II displayed a sustained, non-inactivating current when extracellular application of ACh. ACh-induced currents restored gradually and it took about 60 s to get a complete recovery. ACh-induced current was not affected by extracellular Na(+), but strongly affected by extracellular K(+) and Ca(2+). Depletion of the intracellular Ca(2+) stores by intracellular application of inositol 1,4,5-trisphosphate (IP3) or blocking of the release of intracellular Ca(2+) stores by intracellular application of heparin failed to inhibit this current. ACh-induced currents were inhibited by nifedipine, Cd(2+), tetraethylammonium (TEA), charybdotoxin (CTX), iberiotoxin (IBTX), atropine and d-tubocurarine (DTC), respectively, but not by apamin. In conclusion, ACh stimulates a large conductance, Ca(2+)-activated K(+) current (BK) in guinea pig VHCs II by activation of the influx of Ca(2+) ions, which is mediated by an ACh receptor that could not be defined to be the odd-number muscarinic receptor.