The mechanism of NH + 4 transport in inner medulla is not known. The purpose of these experiments was to study the process that is involved in ammonium (NH + 4 ) transport in cultured inner medullary collecting duct (mIMCD-3) cells. Cells grown on coverslips were exposed to NH + 4 and monitored for pH i changes by the use of the pH-sensitive dye BCECF. The rate of cell acidification following the initial cell alkalinization was measured as an index of NH + 4 transport. The rate of NH + 4 transport was the same in the presence or absence of sodium in the media (0.052 ± 0.003 vs 0.048 ± 0.004 pH/min,P > 0.05), indicating that NH + 4 entry into the cells was independent of sodium. The presence of ouabain, bumetanide, amiloride, barium, or 4.4 -di-isothiocyanostilbene-2-2 -disulfonic acid (DIDS) did not block the NH + 4 -induced cell acidification, indicating lack of involvement of Na + :K + -ATPase, Na + :2Cl - transport, Na + :H + exchange, K + channel, or CI - /base exchange, respectively, in NH + 4 transport. The NH + 4 induced cell acidification was significantly inhibited in the presence of high external [K + ] as compared to low external [K + ] (0.018 ± 0.001 vs. 0.049 ± 0.003 pH/min for 140 mM K + vs. 1.8 mM K + in the media, respectively, P < 0.001). Inducing K + efflux by imposing an outward K + gradient caused intracellular acidification by ∼ 0.3 pH unit in the presence but not the absence of NH + 4 . This K + efflux-induced NH + 4 entry increased by extracellular NH + 4 in a saturable manner with a Km of ∼ 5 mM, blocked by increasing extracellular K + and was not inhibited by barium. The K + efflux-coupled NH + 4 entry was electronueutral as monitored by the use of cell membrane potential probe 3,3 -dipropylthiadicarbocyanine. Thee results are consistent with the exchange of internal K + with external NH + 4 in a 1:1 ratio. The K + -NH + 4 antiporter was inhibited by verapamil and Schering 28080 in a dose-dependent manner, was able to work in reverse mode, and did not show any affinity for H + as a substrate, indicating that it is distinct from other NH + 4 -carrying transporters.We conclude that a unique transporter, a potassium-ammonium (K + /NH + 4 ) antiport, is responsible for NH + 4 transport in renal inner medullary collecting duct cells. This antiporter is sensitive to verapamil and Schering 28080, is electroneutral, and is selective for NH + 4 and K + as substrates. The K + /NH + 4 antiporter may play a significant role in acid-base regulation by excretion of ammonium and elimination of acid.