The renal medullary thick ascending limb (MTAL) of the rat absorbs bicarbonate through luminal H + secretion and basolateral HCO3 transport into the peritubular space. To characterize HCO3 transport, intracellular pH (pHi) was monitored by use of the pH-sensitive fluorescent probe (2',7')-bis-(carboxyethyl)-(5,6)-carboxyfluorescein in fresh suspensions of rat MTAL tubules. When cells were preincubated in HCO3 /C02-containing solutions and then abruptly diluted into HCO3/ C02-free media, the pHi response was an initial alkalinization due to CO2 efflux, followed by an acidification (pHi recovery). The pHi recovery required intracellular HCO3, was inhibited by i0' M diisothiocyanostilbene-2-2'-disulphonic acid (DIDS), and was not dependent on Cl -or Na+. As assessed by use of the cell membrane potential-sensitive fluorescent probe 3,3'-dipropylthiadicarbocyanine, cell depolarization by abrupt Cl -removal from or addition of 2 mM barium into the external medium did not affect HCO3-dependent pHi recovery, and the latter was not associated per se with any change in potential difference, which indicated that HCO3 transport was electroneutral. The HCO3-dependent pHi recovery was inhibited by raising extracellular potassium concentration and by intracellular potassium depletion.