The relevance of nongenomic pathways to regulation of epithelial function by aldosterone is poorly understood. Recently we demonstrated that aldosterone inhibits transepithelial HCO₃^- absorption in the renal medullary thick ascending limb (MTAL) through a nongenomic pathway. Here we examined the transport mechanism(s) responsible for this regulation, focusing on Na⁺/H⁺ exchangers (NHE). In the MTAL, apical NHE3 mediates H⁺ secretion necessary for HCO₃^- absorption; basolateral NHE1 influences HCO₃^- absorption by regulating apical NHE3 activity. In microperfused rat MTALs, addition of 1 nM aldosterone rapidly decreased HCO₃^- absorption by 30%. This inhibition was unaffected by three maneuvers that inhibit basolateral Na⁺/H⁺ exchange and was preserved in MTALs from NHE1 knockout mice, ruling out the involvement of NHE1. In contrast, exposure to aldosterone for 15 min caused a 30% decrease in apical Na⁺/H⁺ exchange activity over the pH_i range 6.5 to 7.7, due to a decrease in V_max. Inhibition of HCO₃^- absorption by aldosterone was not affected by 0.1 mM lumen Zn²⁺ or 1 mM lumen DIDS, arguing against the involvement of an apical H⁺ conductance or apical K⁺-HCO₃^- cotransport. These results demonstrate that aldosterone inhibits HCO₃^- absorption in the MTAL through inhibition of apical NHE3, and identify NHE3 as a target for nongenomic regulation by aldosterone. Aldosterone may influence a broad range of epithelial transport functions important for extracellular fluid volume and acid-base homeostasis through direct regulation of this exchanger.