The epithelial cells of Claudius’ cells form boundary separating endolymph from perilymph in the cochlea and are expected to transport Na+ out of endolymph via epithelial Na+ channel (ENaC). It has been reported in many other epithelia that extracellular ATP decreases ENaC activity through P2Y purinergic signaling by depletion of phosphatidylinositol 4,5-biphosphate, PI(4,5)P2, in the plasma membrane (Lehrmann et al., 2002; Leipziger, 2003). Therefore, we investigated P2Y signaling as a possible regulatory mechanism of ENaC in gerbil Claudius’ cells using voltage-sensitive vibrating probe technique. Gerbils (3-4 weeks old) were anesthetized with sodium pentobarbital (50 – 100 mg/kg; i.p.) and sacrificed to remove temporal bones. The methods for voltage-sensitive vibrating probe have been previously described (Lee and Marcus, 2003). Results showed that uridine triphosphate (UTP) induced partial inhibition of the amiloride-sensitive short-circuit current (Isc), but not with pre-treatment of amiloride. We introduced the antagonists of purinergic receptors to characterize the subtype of UTP-responsive P2Y receptor and compared the effects of UTP on Na+ absorption in the absence and presence of each antagonist. The short circuit current (Isc) was reduced from -9.2±0.5μA/cm2 to -5.6±1.1μA/cm2 in the absence of 100μM reactive blue 2 (n=5), and from -8.5±0.6μA/cm2 to -8.0±0.5μA/cm2 in the presence of reactive blue 2 (n=5). In contrast, neither 100 μM suramin nor 100 μM pyridoxalphosphate-6-azophenyl-2′, 4′-disulfonic acid (PPADS) had any significant effect on the action of UTP. Isc was reduced from -10.0±1.3μA/cm2 to -7.0±1.3μA/cm2 in the absence of suramin (n=5), and from -10.0±1.2μA/cm2 to -7.9±1.3μA/cm2 in the presence of suramin (n=5). Isc was reduced from -9.2±0.7μA/cm2 to -6.1±0.6μA/cm2 in the absence of PPADS, n=6), and from -8.9±0.9μA/cm2 to -5.5±0.7μA/cm2 in the presence of PPADS (n=6). These results indicate this P2Y receptor as the P2Y4 subtype. Then, we investigated the role of PLC signaling cascade in the regulation of ENaC activity by UTP. The inhibition of Isc by UTP was significantly reduced in the presence of PLC inhibitors, U73122 (10μM) and edelfosine (10μM). Isc was reduced from -10.2±1.0μA/cm2 to -6.0±1.1μA/cm2 in the absence of U73122 (n=5), and from -9.2±1.1μA/cm2 to -8.8±1.1μA/cm2 in the presence of U73122 (n=5). Isc was reduced from -9.1±1.2μA/cm2 to -5.7±1.5μA/cm2 in the absence of edelfosine (n=5), and from -8.3±1.3μA/cm2 to -7.7±1.4μA/cm2 in the presence of edelfosine (n=5). These results suggest that the physiological role of P2Y4 receptor in Claudius’ cells likely to regulate Na+ homeostasis in the endolymph. The acute inhibition of ENaC activity by activation of P2Y4 receptor is possibly mediated by decrease of phosphatidylinositol 4,5-biphosphate, PI(4,5)P2, in the plasma membrane through phospholipase C activation.