Several members of the Na-HCO cotransporter (NBC) family have recently been identified functionally and partly characterized, including rkNBC1, NBCn1, and NBC3. Regulation of these NBCs may play a role in the maintenance of intracellular pH and in the regulation of renal acid-base balance. However, it is unknown whether the expressions of these NBCs are regulated in response to changes in acid-base status. We therefore tested whether chronic metabolic acidosis (CMA) affects the abundance of these NBCs in kidneys using two conventional protocols. In protocol 1, rats were treated with NH(4)Cl in their drinking water (12 +/- 1 mmol. rat(-1). day(-1)) for 2 wk with free access to water (n = 8). Semiquantitative immunoblotting demonstrated that whole kidney abundance of NBCn1 and NBC3 in rats with CMA was dramatically increased to 995 +/- 87 and 224 +/- 35%, respectively, of control levels (P < 0.05), whereas whole kidney rkNBC1 was unchanged (88 +/- 14%). In protocol 2, rats were given NH(4)Cl in their food (10 +/- 1 mmol. rat(-1). day(-1)) for 7 days, with a fixed daily water intake (n = 6). Consistent with protocol 1, whole kidney abundances of NBCn1 (262 +/- 42%) and NBC3 (160 +/- 31%) were significantly increased compared with controls (n = 6), whereas whole kidney rkNBC1 was unchanged (84 +/- 17%). In both protocols, immunocytochemistry confirmed upregulation of NBCn1 and NBC3 with no change in the segmental distribution along the nephron. Consistent with the increase in NBCn1, measurements of pH transients in medullary thick ascending limb (mTAL) cells in kidney slices revealed two- to threefold increases in DIDS- sensitive, Na(+)-dependent HCO uptake in rats with CMA. In conclusion, CMA is associated with a marked increase in the abundance of NBCn1 in the mTAL and NBC3 in intercalated cells, whereas the abundance of NBC1 in the proximal tubule was not altered. The increased abundance of NBCn1 may play a role in the reabsorption of NH in the mTAL and increased NBC3 in reabsorbing HCO.