We hypothesized that the function of duodenocyte apical membrane acid-base transporters are essential for H⁺ absorption from the lumen. We thus examined the effect of inhibition of Na⁺/H⁺ exchanger-3 (NHE3), cystic fibrosis transmembrane regulator (CFTR) or apical anion exchangers on transmucosal CO₂ diffusion and HCO₃- secretion in rat duodenum. Duodena were perfused with a pH 6.4 high CO₂ solution or pH 2.2 low CO₂ solution with the NHE3 inhibitor, S3226, the anion transport inhibitor, DIDS, or pretreatment with the potent CFTR inhibitor, CFTR_inh-172, with simultaneous measurements of luminal and portal venous (PV) pH and [CO₂]. Luminal high CO₂ solution increased CO₂ absorption and HCO₃- secretion, accompanied by PV acidification and PV PCO₂ increase. During CO₂ challenge, CFTR_inh-172 induced HCO₃- absorption while inhibiting PV acidification. S3226 reversed CFTRinh-associated HCO3- absorption. Luminal pH 2.2 challenge increased H⁺ and CO₂ absorption and acidified the PV, inhibited by CFTR_inh-172 and DIDS, but not by S3226. CFTR inhibition and DIDS reversed HCO₃- secretion to absorption, and inhibited PV acidification during CO₂ challenge, suggesting that HCO₃- secretion helps facilitate CO₂/H⁺ absorption. Furthermore, CFTR inhibition prevented CO₂-induced cellular acidification, reversed by S3226. Reversal of increased HCO₃- loss by NHE3 inhibition and reduced intracellular acidification during CFTR inhibition is consistent with activation or unmasking of NHE3 activity by CFTR inhibition, increasing cell surface H+ available to neutralize luminal HCO₃- with consequent CO₂ absorption. NHE3, by secreting H⁺ into the luminal microclimate, facilitates net transmucosal H⁺ absorption with a mechanism similar to proximal tubular CO₂ absorption.