Expression of inositol-1,4,5-trisphosphate (IP3) receptor-binding protein (IRBIT) has been reported in epithelial cells. However, its role in pH, regulation is not well understood. In this study, we investigated the role of IRBIT in pH(i) regulation, mediated by Ne+/H+ exchangers (NHEs), in salivary glands. We measured pH(i) recovery from cell acidification in BCECF-loaded salivary HSG cells. Western blot and co-immunoprecipitation (CO-IP) assays were also performed, showing that NHE1, 2 and 3 are expressed, and IRBIT binds to NHE3. HOE642, a specific NHE1 blocker, inhibited pH, recovery, but 40% pH(i) recovery was still observed even at the highest concentration of HOE642. Furthermore, pretreatment of the cells with siIRBIT significantly inhibited pH(i) recovery, indicating that NHE3 potentially plays a role in pH(i) recovery as well. The amount of membrane-localized NHE3 and its interaction with IRBIT are also significantly increased by cell acidification. In addition, we found that Ste20p-related proline alanine-rich kinase (SPAK) reverses the effect of IRBIT on membrane NHE3 translocation. Taken together, we conclude that IRBIT plays an important role in pH(i) regulation, mediated by NHE3, and further regulated by SPAK. (c) 2013 Elsevier Inc. All rights reserved.