The weatherloach, Misgurnus angulliacaudatus, is an intestinal air-breathing, freshwater fish that has the unique ability to excrete ammonia through gut volatilization when branchial and cutaneous routes are compromised during high environmental ammonia or air exposure. We hypothesized that transepithelial gut NH4+ transport is facilitated by an apical Na+/H+ (NH4+) exchanger (NHE) and a basolateral Na+/K+(NH4 +)-ATPase, and that gut boundary layer alkalinization (NH4 + → NH3 + H+) is facilitated by apical HCO3– secretion through a Cl–/HCO3– anion exchanger. This was tested using a pharmacological approach with anterior (digestive) and posterior (respiratory) intestine preparations mounted in pH-stat-equipped Ussing chambers. The anterior intestine had a markedly higher conductance, increased short-circuit current, and greater net base (Jbase) and ammonia excretion rates (Jamm) than the posterior intestine. In the anterior intestine, HCO3– accounted for 70% of Jbase. In the presence of an imposed serosal–mucosal ammonia gradient, inhibitors of both NHE (EIPA, 0.1mmoll–1) and Na+/K+-ATPase (ouabain, 0.1mmoll–1) significantly inhibited Jamm in the anterior intestine, although only EIPA had an effect in the posterior intestine. In addition, the anion exchange inhibitor DIDS significantly reduced Jbase in the anterior intestine although only at a high dose (1mmoll–1). Carbonic anhydrase does not appear to be associated with gut alkalinization under these conditions as ethoxzolamide was without effect on Jbase. Membrane fluidity of the posterior intestine was low, suggesting low permeability, which was also reflected in a lower mucosal–serosal Jamm in the presence of an imposed gradient, in contrast to that in the anterior intestine. To conclude, although the posterior intestine is highly modified for gas exchange, it is the anterior intestine that is the likely site of ammonia excretion and alkalinization leading to ammonia volatilization in the gut.