Zebrafish Na⁺/H⁺ exchanger 3b (zNHE3b) is highly expressed in the apical membrane of ionocytes where Na⁺ is absorbed from ion-poor fresh water against a concentration gradient. Much in vivo data indicated that zNHE3b is involved in Na⁺ absorption but not leakage. However, zNHE3b-mediated Na⁺ absorption has not been thermodynamically explained, and zNHE3b activity has not been measured. To address this issue, we overexpressed zNHE3b in Xenopus oocytes and characterized its activity by electrophysiology. Exposure of zNHE3b oocytes to Na⁺-free media resulted in significant decrease in intracellular pH (pH_i) and intracellular Na⁺ activity (aNa_i). aNa_i increased significantly when the cytoplasm was acidified by media containing CO₂/HCO₃^- or butyrate. Activity of zNHE3b was inhibited by amiloride or 5-ethylisopropyl amiloride (EIPA). Although the activity was accompanied by a large hyperpolarization of ~50 mV, voltage clamp experiments showed that Na⁺/H⁺-exchange activity of zNHE3b is electroneutral. Exposure of zNHE3b oocytes to medium containing NH₃/NH₄⁺ resulted in significant decreases in pH_i and aNa_i and significant increase in intracellular NH₄⁺ activity, indicating that zNHE3b mediates the Na⁺/NH₄⁺ exchange. In low-Na⁺ (0.5 mM) media, zNHE3b oocytes maintained intracellular Na⁺ activity of 1.3 mM, and Na⁺-influx was observed when intracellular pH was decreased by media containing CO₂/HCO₃^- or butyrate. These results provide thermodynamic evidence that zNHE3b mediates Na⁺ absorption from ion-poor fresh water by its Na⁺/H⁺ and Na⁺/NH₄⁺-exchange activities.