To characterize NH4+ transport in the renal medullary thick ascending limb (MTAL), cell pH was monitored with the use of 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein in suspensions of rat MTAL tubules in CO2-free media. Abrupt exposure to NH4Cl led to initial cell alkalinization (NH3 entry) followed by cell acidification due to NH4+ influx. The latter had 1 microM amiloride- and barium-sensitive components; the barium effect was unchanged when K+ conductances were completely blocked by quinidine, as assessed with the use of the cell membrane potential-sensitive fluorescent probe 3,3'-dipropylthiadicarbocyanine. NH4+ entry-induced depolarization was abolished by 1 microM amiloride, but it was unaffected by barium. In NH(4+)-free media, barium, verapamil, and raising of the extracellular K+ concentration alkalinized MTAL cells; imposing an outward directed K+ gradient in Na(+)-free medium induced cellular acidification, which was abolished by barium and verapamil but not by other K+ channel and Na+/H+ antiport inhibitors (quinidine and 2 mM amiloride). As measured with a K(+)-selective extracellular electrode, a component of K+ efflux (in presence of furosemide, ouabain, and quinidine) was stimulated by decreasing the extracellular pH from 7.4 to 7.0 and inhibited by barium and verapamil. It was also demonstrated that the K+/H+ antiporter transports NH4+ better than H+ at physiological NH4+ and H+ concentrations. These results demonstrate the presence in MTAL cells of two novel NH4+ transport pathways, amiloride-sensitive NH4+ conductance and barium- and verapamil-sensitive K+/NH4+(H+) antiport.