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⁺ ( pHi : 0.88±0.11 pH units, n=10). Subsequently the basolateral perfusion solution was changed to CO₂/HCO₃^- buffer with and without Na⁺. Basolateral NHE 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 vs. 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 immunolabeling experiments.RT-PCR analysis revealed no evidence for NBC4, NCBE or 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.