Although aldosterone influences a variety of cellular processes through nongenomic mechanisms, the significance of nongenomic pathways for aldosterone-induced regulation of epithelial function is not understood. Recently we demonstrated that aldosterone inhibits transepithelial HCO₃^- absorption in the medullary thick ascending limb (MTAL) through a nongenomic pathway. This inhibition is mediated through a direct cellular action of aldosterone to inhibit the apical membrane NHE3 Na⁺/H⁺ exchanger. The present study was designed to identify the intracellular signaling pathway(s) responsible for this aldosterone-induced transport regulation. In rat MTALs perfused in vitro, addition of 1 nM aldosterone to the bath decreased HCO₃^- absorption by 30%. This inhibition was not mediated by cAMP/PKA and was not prevented by inhibitors of PKC or PI3-K, pertussis toxin, or rapamycin. The inhibition of HCO₃^- absorption by aldosterone was largely eliminated by the MEK/ERK inhibitors U0126 and PD98059. Aldosterone increased ERK activity 1.8-fold in microdissected MTALs. This ERK activation is rapid ( 5 min) and is blocked by U0126 or PD98059, but is unaffected by spironolactone or actinomycin D. Pretreatment with U0126 to block ERK activation prevented the effect of aldosterone to inhibit apical NHE3. These data demonstrate that aldosterone inhibits NHE3 and HCO₃^- absorption in the MTAL through rapid activation of the ERK signaling pathway. The results identify NHE3 as a target for nongenomic regulation by aldosterone and establish a role for ERK in the acute regulation of NHE3 and its epithelial absorptive functions.