Mechanisms involved in basolateral HCO transport were examined in the in vitro microperfused rat medullary thick ascending limb of Henle (MTALH) by microfluorometric monitoring of cell pH. Removing peritubular Cl(-) induced a cellular alkalinization that was inhibited in the presence of peritubular 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) and blunted in the absence of external CO(2)/HCO. The alkalinization elicited by removing peritubular Cl(-) persisted in the bilateral absence of Na(+), together with a voltage clamp. When studied in Cl(-)-free solutions, lowering peritubular pH induced a base efflux that was inhibited by peritubular DIDS or by the absence of external CO(2)/HCO. Removing peritubular Na(+) elicited a cellular acidification that was accounted for by stimulation of a DIDS- and ethylisopropylamiloride (EIPA)-insensitive Na(+)-HCO cotransport and inhibition of a basolateral Na(+)/H(+) exchange. Increasing bath K(+) induced an intracellular alkalinization that was inhibited in the absence of external CO(2)/HCO. At 2 mM, peritubular Ba(2+), which inhibits the K(+)-Cl(-) cotransport, did not induce any change in transepithelial voltage but elicited a cellular alkalinization and inhibited K(+)-induced cellular alkalinization, consistent with the presence of a basolateral, electroneutral Ba(2+)-sensitive K(+)-Cl(-) cotransport that may operate as a K(+)-HCO cotransport. This cotransport was inhibited in the peritubular presence of furosemide, [(dihydroindenyl)oxy]alkanoic acid, 5-nitro-2-(3-phenylpropylamino)benzoate, or DIDS. At least three distinct basolateral HCO transport mechanisms are functional under physiological conditions: electroneutral Cl(-)/HCO exchange, DIDS- and EIPA-insensitive Na(+)-HCO cotransport, and Ba(2+)-sensitive electroneutral K(+)-Cl(-)(HCO) cotransport.