Apical membrane Na+/H+ exchange mediates virtually all of transepithelial HCO3- absorption in the rat medullary thick ascending limb (MTAL). Regulation of the apical exchanger by intracellular pH (pHi) and hyperosmolality was studied in the isolated, perfused MTAL by measurement of pHi using the fluorescent probe 2‘,7‘-bis-(carboxyethyl)-5,6-carboxyfluorescein. Under isosmotic conditions (290 mosmol/kg H2O), the Na+/H+ exchange rate increased sigmoidally over the pHi range 7.8 to 6.5 (Hill coefficient = 2.1), consistent with cooperative activation of the exchanger by internal H+. The exchanger had a high apparent affinity for intracellular H+ (apparent pK = 7.36), which resulted in the exchanger being maximally active at resting pHi and insensitive to changes in pHi over the physiologic pHi range (6.5-7.2). Hyperosmolality (590 mosmol/kg H2O) inhibited Na+/H+ exchange by at least 35% at all pHi values studied and induced pHi dependence of the exchanger between 6.5 and 7.2. The inhibition by hyperosmolality appeared to be the result of an acid shift of the pHi dependence curve of the exchanger. These functional properties of apical membrane Na+/H+ exchange can account for our previous observations that hyperosmolality inhibited net HCO3- absorption and that the rate of HCO3- absorption did not correlate with pHi. Apical membrane Na+/H+ exchange in the MTAL differs functionally from Na+/H+ exchange in other cell types in which exchanger activity is stimulated rather than inhibited by hyperosmolality.