In this study, a scanning ion-selective electrode technique (SIET) was applied to measure H(+), Na(+), and NH(4)(+) gradients and apparent fluxes at specific cells on the skin of medaka larvae. Na(+) uptake and NH(3)/NH(4)(+) excretion were detected at most mitochondrion-rich cells (MRCs). H(+) probing at MRCs revealed two group of MRCs, i.e., acid-secreting and base-secreting MRCs. Treatment with EIPA (100 muM) blocked 35% of the NH(3)/NH(4)(+) secretion and 54% of the Na(+) uptake, suggesting that the Na(+)/H(+) exchanger (NHE) is involved in Na(+) and NH(3)/NH(4)(+) transport. Low-Na(+) water (<0.001 mM) or high-NH(4)(+) (5 mM) acclimation simultaneously increased Na(+) uptake and NH(3)/NH(4)(+) excretion but decreased or even reversed the H(+) gradient at the skin and MRCs. The correlation between NH(4)(+) production and H(+) consumption at the skin surface suggests that MRCs excrete nonionic NH(3) (base) by an acid-trapping mechanism. Raising the external NH(4)(+) significantly blocked NH(3)/NH(4)(+) excretion and Na(+) uptake. In contrast, raising the acidity of the water (pH 7 to pH 6) enhanced NH(3)/NH(4)(+) excretion and Na(+) uptake by MRCs. In situ hybridization and real-time PCR showed that the mRNAs of the Na(+)/H(+) exchanger (slc9a3) and Rhesus glycoproteins (Rhcg1 and Rhbg) were colocalized in MRCs of medaka, and their expressions were induced by low-Na(+) acclimation. This study suggests a novel Na(+)/NH(4)(+) exchange pathway in apical membranes of MRCs, in which a coupled NHE and Rh glycoprotein is involved and the Rh glycoprotein may drive the NHE by generating H(+) gradients across apical membranes of MRCs.