The choroid plexus epithelium (CPE) secretes the major fraction of the cerebrospinal fluid (CSF). The Na(+)-HCO(3)(-) transporter Ncbe/Nbcn2 in the basolateral membrane of CPE cells is important for Na(+)-dependent pH(i) increases and probably for CSF secretion. In the current study, the anion transport inhibitor DIDS had no effect on the residual pH(i) recovery in acidified CPE from Ncbe/Nbcn2 knockout mouse by 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF)-fluorescence microscopy in the presence of CO(2)/HCO(3)(-) (Ncbe/Nbcn2-ko+DIDS 109% of control, P = 0.76, n = 5). Thus Ncbe/Nbcn2 mediates the DIDS-sensitive Na(+)-dependent pH(i) recovery in the CPE. The Na(+)/H(+) exchanger-1 Nhe1 is proposed to mediate similar functions as Ncbe/Nbcn2 in CPE. Here, we immunolocalize the Nhe1 protein to the luminal membrane domain in mouse and human CPE. The Na(+)-dependent pH(i) recovery of Nhe1 wild-type (Nhe1-wt) mice in the absence of CO(2)/HCO(3)(-) was abolished in the Nhe1 knockout CPE (Nhe1-ko 0.37% of Nhe1-wt, P = 0.0007, n = 5). In Ncbe/Nbcn2-ko mice, Nhe1 was targeted to the basolateral membrane. Nevertheless, the luminal Na(+)-dependent pH(i) recovery was increased in Ncbe/Nbcn2-ko compared with wild-type littermates (Nhe1-ko 146% of Nhe1-wt, P = 0.007, n = 5). Whereas the luminal Nhe activity was inhibited by the Nhe blocker EIPA (10 microM) in the Ncbe/Nbcn2-wt, it was insensitive to the inhibitor in Ncbe/Nbcn2-ko (Ncbe/Nbcn2-ko+EIPA 100% of control, P = 0.98, n = 5). This indicates that a luminal EIPA-insensitive Nhe was induced in Ncbe/Nbcn2-ko CPE and that EIPA-sensitive Nhe activity was basolateral. The Nhe1 translocation in Ncbe/Nbcn2-ko CPE may reflect a compensatory response, which provides the cells with better means of regulating pH(i) or transporting Na(+) after Ncbe/Nbcn2 disruption.