The electroneutral, Na⁺-coupled HCO₃⁻ co-transporter (NBCn1) is expressed in the basolateral membrane of rat medullary thick ascending limb (mTAL) segments and is up-regulated strongly during metabolic acidosis. We have suggested previously that NBCn1-mediated HCO₃⁻ uptake across the basolateral cell membrane is an important buffering mechanism during the transcellular transport of NH₄⁺ in the mTAL. To investigate this further we treated rats with a low-K⁺ diet for 4 days, a manoeuvre known to increase proximal tubular ammoniagenesis and delivery of luminal NH₄⁺ to the mTAL. Hypokalaemia strongly increased immunolabelling of basolateral NBCn1 in the mTAL of the inner stripe of the outer medulla. Immunoblotting revealed an eightfold up-regulation of NBCn1 protein in K⁺-depleted rats. Subsequently we used in vitro perfusion of isolated mTAL and BCECF imaging to investigate Na⁺-coupled HCO₃⁻ influx in normal and K⁺-depleted rats. Hypokalaemia induced a threefold up-regulation of Na⁺-coupled HCO₃⁻ influx (1.64±0.28 vs. 0.47±0.05 pH units/minute, n=8). The Na⁺-dependent alkalinization was also significantly larger in K⁺-depleted rats (0.38±0.04 vs. 0.23±0.03 pH units). These data indicate that K⁺-depleted rats respond with a strong up-regulation of NBCn1 protein and function, probably to cope with the higher tubular load of NH₄⁺, and strengthen our previous suggestion that NBCn1 is an important player in the excretion of NH₄⁺. They also indicate that it is the delivery of luminal NH₄⁺ rather than systemic changes of pH that determine the expression of NBCn1.