NH4/+/NH3 fluxes were used to probe apical Na-K-2Cl transport activity of macula densa (MD) cells from rabbit kidney. In the presence of 25 mM NaCl and 5 mM Ba2+, addition of 20 mM NH4/+ to the lumen produced a profound intracellular acidification, and ~80% of the initial acidification rate was bumetanide sensitive. The NH4/+-induced acidification rate was dependent on luminal Cl- and Na+ with apparent affinities of 17 ± 4 mM (Hill number 1.45) and 1.0 ± 0.3 mM, respectively. In the presence of saturating luminal NaCl concentration ([NaCl](L)), blockade of basolateral Cl- efflux with 10 μM 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB) reduced the NH4/+-induced acidification rate by 51 ± 6% (P > 0.01, n = 5). Under similar conditions, dibutyryl-cAMP (DBcAMP) + forskolin increased the NH4/+-induced acidification rate by 27%, whereas it produced no detectable effect at low luminal NaCl concentration. Most of the observed DBcAMP + forskolin effect was probably due to the stimulation of the basolateral Cl- conductance, since, in the presence of basolateral NPPB, this activation was changed to a 17.1% and 16.6% inhibition of the NH4/+-induced acidification rate observed at high or low [NaCl](L), respectively. We conclude that the cotransporter found in MD cells displays, with respect to other Na-K-2Cl cotransporters, a relatively high affinity for luminal Na+ and luminal Cl- and can be specifically inhibited by increases in intracellular Cl- and cAMP concentrations.