Several studies suggest the involvement of Na+ and HCO3- transport in the formation of cerebrospinal fluid. Two Na+-dependent HCO3- transporters were recently localized to the epithelial cells of the rat choroid plexus (NBCn1 and NCBE), and the mRNA for a third protein was also detected (NBCe2) (Praetorius J, Nejsum LN, and Nielsen S. Am J Physiol Cell Physiol 286: C601-C610, 2004). Our goal was to immunolocalize the NBCe2 to the choroid plexus by immunohistochemistry and immunogold electronmicroscopy and to functionally characterize the bicarbonate transport in the isolated rat choroid plexus by measurements of intracellular pH (pHi) using a dual-excitation wavelength pH-sensitive dye (BCECF). Both antisera derived from COOH-terminal and NH2-terminal NBCe2 peptides localized NBCe2 to the brush-border membrane domain of choroid plexus epithelial cells. Steady-state pHi in choroidal cells increased from 7.03 +/- 0.02 to 7.38 +/- 0.02 (n=41) after addition of CO2/HCO3- into the bath solution. This increase was Na+ dependent and inhibited by the Cl- and HCO3- transport inhibitor DIDS (200 muM). This suggests the presence of Na+-dependent, partially DIDS-sensitive HCO3- uptake. The pHi recovery after acid loading revealed an initial Na+ and HCO3- -dependent net base flux of 0.828 +/- 0.116 mM/s (n = 8). The initial flux in the presence of CO2/HCO3- was unaffected by DIDS. Our data support the existence of both DIDS-sensitive and -insensitive Na+- and HCO3- -dependent base loader uptake into the rat choroid plexus epithelial cells. This is consistent with the localization of the three base transporters NBCn1, Na+-driven Cl- bicarbonate exchanger, and NBCe2 in this tissue.