The proper homeostasis of the liquid lining the surface of the middle ear cavity is vitally important for maintaining a fluid-free middle ear cavity. Disruption of this homeostasis leads to fluid collection in the middle ear cavity and results in otitis media with effusion. We demonstrated the molecular and functional expression of the Na+/H+ exchanger (NHE)s in normal human middle ear epithelial (NHMEE) cells. We also evaluated the role of NHEs in fluid absorption and the effect of dexamethasone on NHE function and NHE-dependent fluid absorption in NHMEE cells. Western blot analysis was performed for NHE1, -2, and -3 in NHMEE cells. The fluid absorption rate was measured after liquid application on the luminal surface of the cells. Intracellular pH (pHi) was measured using the pH-sensitive fluorescent probe bis-(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF)-AM. NHE activity was determined as Na+-induced pHi recovery from an acid load achieved by luminal exposure to 40 mmol/l NH4Cl. NHE1, -2 and -3 were all expressed in the NHMEE cells. The pHi recovery rate was suppressed by inhibition of NHE2 and -3 with HOE694 at concentrations greater than 50 microM. Inhibition of NHE3 with 650 microM of HOE694 or S3226 significantly decreased the fluid absorption rate. Dexamethasone increased the Na+-induced pHi recovery rate which was reversed by the inhibition of NHE3 with 650 microM of HOE694. Dexamethasone treatment up-regulated NHE3 expression in a dose-dependent manner. The fluid absorption rate was increased by treatment with dexamethasone (10(-7) M) and reversed by the inhibition of NHE3. In summary, we have shown that NHE3 are involved in the regulation of both pHi and fluid absorption on the luminal surface of NHMEE cells. Dexamethasone stimulates NHE3 expression and NHE3-dependent fluid absorption in NHMEE cells. These findings provide a new insight into mechanisms that regulate periciliary fluid and the therapeutic mechanisms behind steroid treatment of otitis media with effusion.