1. We have used highly purified right-side-out luminal and basolateral membrane vesicles (LMVs and BLMVs) isolated from rat medullary thick ascending limb (MTAL) to study directly the possible roles of the LMV and BLMV Na(+)-H+ exchangers in the transport of NH4+. 2. Extravesicular NH4+ ((NH4+)o) inhibited outward H+ gradient-stimulated 22Na+ uptake in both types of vesicles. This inhibition could not be accounted for by alteration of intravesicular pH (pHi). 3. Conversely, in both plasma membrane preparations, the imposition of outward NH4+ gradients stimulated 22Na+ uptake at the acidic pHi (6.60) of MTAL cells, under conditions in which possible alterations in pHi were prevented. All NH4+ gradient-stimulated Na+ uptake was sensitive to 0.5 mM 5-(N,N-dimethyl)-amiloride. 4. The BLMV and LMV Na(+)-H+ exchangers had a similar apparent affinity for internal H+ (Hi+), with pK (-log of dissociation constant) values of 6.58 and 6.52, respectively. 5. These findings indicate that NH4+ interacts with the external and internal transport sites of the LMV and BLMV Na(+)-H+ antiporters, and that both of these exchangers can mediate the exchange of internal NH4+ ((NH4+)i) for external Na+ (Na+o) at the prevailing pHi of MTAL cells. 6. We conclude that operation of the BLMV Na(+)-H+ exchanger on the NH4(+)-Na+ mode may represent an important pathway for mediating the final step of NH4+ absorption, whereas transport of NH4+ on the apical antiporter may provide negative feedback regulation of NH4+ absorption.