The electroneutral Na⁺‐dependent HCO₃⁻ transporter NBCn1 is strongly expressed in the basolateral membrane of rat medullary thick ascending limb cells (mTAL) and is up‐regulated during NH₄⁺‐induced metabolic acidosis. Here we used in vitro perfusion and BCECF video‐imaging of mTAL tubules to investigate functional localization and regulation of Na⁺‐dependent HCO₃⁻ influx during NH₄⁺‐induced metabolic acidosis. Tubule acidification was induced by removing luminal Na⁺ (ΔpH_i: 0.88 ± 0.11 pH units, n= 10). Subsequently the basolateral perfusion solution was changed to CO₂/HCO₃⁻ buffer with and without Na⁺. Basolateral Na⁺–H⁺ exchange function was inhibited with amiloride. Na⁺‐dependent HCO₃⁻ influx was determined by calculating initial base flux of Na⁺‐mediated re‐alkalinization. In untreated animals base flux was 8.4 ± 0.9 pmol min⁻¹ mm⁻¹. A 2.4‐fold increase of base flux to 21.8 ± 3.2 pmol min⁻¹ mm⁻¹ was measured in NH₄⁺‐treated animals (11 days, n= 11). Na⁺‐dependent re‐alkalinization was significantly larger when compared to control animals (0.38 ± 0.03 versus 0.22 ± 0.02 pH units, n= 10). In addition, Na⁺‐dependent HCO₃⁻ influx was of similar magnitude in chloride‐free medium and also up‐regulated after NH₄⁺ loading. Na⁺‐dependent HCO₃⁻ influx was not inhibited by 400 μm DIDS. A strong up‐regulation of NBCn1 staining was confirmed in immunolabelling experiments. RT‐PCR analysis revealed no evidence for the Na⁺‐dependent HCO₃⁻ transporter NBC4 or the two Na⁺‐dependent CI⁻/HCO₃⁻ exchangers NCBE and NDCBE. These data strongly indicate that rat mTAL tubules functionally express basolateral DIDS‐insensitive NBCn1. Function and protein are strongly up‐regulated during NH₄⁺‐induced metabolic acidosis. We suggest that NBCn1‐mediated basolateral HCO₃⁻ influx is important for basolateral NH₃ exit and thus NH₄⁺ excretion by means of setting pH_i to a more alkaline value.