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 CO₂/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 CO₂/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 CO₂/HCO. At 2 mM, peritubular Ba²⁺, 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 Ba²⁺-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 Ba²⁺-sensitive electroneutral K⁺-Cl⁻(HCO) cotransport.