T he regulation of epithelial Na⁺/H⁺ exchangers (NHEs) by hyposmolality is poorly understood. In the renal medullary thick ascending limb (MTAL), transepithelial bicarbonate (HCO₃^–) absorption is mediated by apical membrane Na⁺/H⁺ exchange, attributable to NHE3. In the present study we examined the effects of hyposmolality on apical Na⁺/H⁺ exchange activity and HCO₃^– absorption in the MTAL of the rat. In MTAL perfused in vitro with 25 mM HCO₃^– solutions, decreasing osmolality in the lumen and bath by removal of either mannitol or sodium chloride significantly increased HCO₃^– absorption. The responses to lumen addition of the inhibitors ethylisopropyl amiloride, amiloride, or HOE 694 are consistent with hyposmotic stimulation of apical NHE3 activity and provide no evidence for a role for apical NHE2 in HCO₃^– absorption. Hyposmolality increased apical Na⁺/H⁺ exchange activity over the pH_i range 6.5–7.5 due to an increase in V_max. Pretreatment with either tyrosine kinase inhibitors or with the tyrosine phosphatase inhibitor molybdate completely blocked stimulation of HCO₃^– absorption by hyposmolality. These results demonstrate that hyposmolality increases HCO₃^– absorption in the MTAL through a novel stimulation of apical membrane Na⁺/H⁺ exchange and provide the first evidence that NHE3 is regulated by hyposmotic stress. Stimulation of apical Na⁺/H⁺ exchange activity in renal cells by a decrease in osmolality may contribute to such pathophysiological processes as urine acidification by diuretics, diuretic resistance, and renal sodium retention in edematous states.J. Clin. Invest. 104:1593–1602 (1999).