Furosemide is a loop diuretic that inhibits NaCl reabsorption in the thick ascending limb (TAL). In addition, furosemide causes urinary acidification and eventually metabolic alkalosis. It is traditionally explained by an increased Na⁺ load to the distal tubule, which is suggested to facilitate H⁺ secretion via the apical H⁺-ATPase in α-intercalated cells. The direct role of the thick ascending limb on the urinary acidification, however, has never been investigated. Here we measured pH_i in single perfused mouse mTALs with BCECF-AM. Interestingly, luminal furosemide (100 µM) caused a major, stable and reversible intracellular alkalinization both in HEPES (0.33 ± 0.04, n=7) and CO₂/HCO₃^--buffered conditions (0.14 ± 0.03, n=5). This alkalinization likely indicates increased H⁺ excretion from the mTAL cytosol. Intriguingly, the furosemide-induced alkalinization was completely blocked by 1 mM luminal amiloride that fully inhibits the apical NHE3 antiporter. Thus, furosemide likely causes a NHE3-dependent secretion of H⁺ to the lumen. To investigate this, we measured the pH_o of the tubular lumen with BCECF acid. Furosemide indeed caused a reversible luminal acidification from pH 6.92 ± 0.04 to 6.46 ± 0.03 (n=5) providing direct evidence for this suggested mechanism. In contrast, luminal amiloride alkalinized the lumen providing again direct evidence for an NHE-dependent mechanism of urinary acidification. These results revise the mechanistic understanding of furosemide-induced urinary acidification.