The acute effect of Angiotensin Converting Enzyme inhibition (ACEi) on proximal convoluted tubule (PCT) function is well documented. However, the effect of chronic treatment is less known. The aim of this work was to evaluate the effect of chronic ACEi on PCT acidification (J_HCO3-). Rats received enalapril (10 mg / kg^-1 /day^-1, added to the drinking water) during three months. Micropuncture experiments were performed to measure the effect of chronic ACEi on J_HCO3-. Nitric oxide (NO^.) synthesis in kidney cortex homogenates was assessed by quantifying the conversion of [¹⁴C]-L-arginine to [¹⁴C]-L-citrulline. Western blot analysis was utilized to determine the abundances of V-H⁺ATPase and NHE3 isoform of the Na⁺/H⁺ exchanger in proximal brush border membrane vesicles (BBMV). Enalapril treatment induced a ~50% increase in J_HCO3-. Luminal perfusion with Ethyl-Isopropyl Amiloride (EIPA) 10^-4M or Bafilomycin 10^-6M decreased J_HCO3- by ~60% and ~30% respectively, in both control and enalapril-treated rats. The effect of EIPA and Bafilomycin on absolute J^HCO3- was larger in enalapril-treated than in control rats. Acute inhibition of NO^. synthesis with NG nitro-L-arginine methyl esther abolished the enalapril-induced increase in J_HCO3-. Cortex homogenates from enalapril-treated rats displayed a 46% increase in nitric oxide synthase (NOS) activity when compared to those from untreated animals. Enalapril treatment did not affect the abundances of NHE3 and V-H⁺ATPase in BBMV. Our results suggest that PCT acidification is increased during chronic ACEi probably due to an increase in NO^.synthesis, which would stimulate Na⁺/H⁺ exchange and electrogenic proton transport.