We studied the opening mechanism of Ca2+-permeable, channels formed with mouse transient receptor potential type 5 (mTRP5) using Xenopus oocytes. After stimulation of coexpressed muscarinic M, receptors with acetylcholine (ACh) in a Ca2+-free solution, switching to 2 mM Ca2+-containing solution evoked a large Cl- current, which reflects the opening of endogenous Ca2+-dependent Cl- channels following Ca2+ entry through the expressed channels. The ACh-evoked response was not affected by a depletion of Ca2+ store with thapsigargin but was inhibited by preinjection of antisense oligodeoxynucleotides (ODNs) to G(q), G(11), or both. The mTRP5 channel response was also induced by a direct activation of G proteins with injection of guanosine 5'-3-O-(thio)tri phosphate (GTP gammaS). The ACh- and GTP gammaS-evoked responses were inhibited by either pretreatment with a phospholipase C inhibitor, U73122, or an inositol-1,4,5-trisphosphate (IP3) receptor inhibitor, xestospongin C (XeC). An activation of IP3 receptors with injection of adenophostin A (AdA) evoked the mTRP5 channel response in a dose-dependent manner. The AdA-evoked response was not suppressed by preinjection of antisense ODNs to G(q/11) or U73122 but was suppressed by either preinjection of XeC or a peptide mimicking the IP, binding domain of Xenopus IP3 receptor. These findings suggest that the activation of IP3 receptor is essential for the opening of mTRP5 channels, and that neither G proteins, phosphoinositide metabolism, nor depletion of the Ca2+ store directly modifies the IP3 receptor-linked opening of mTRP5 channels.