T o study the roles of Na⁺-dependent H⁺ transporters, we characterized H⁺ efflux mechanisms in the pancreatic duct in wild-type, NHE2^–/–, and NHE3^–/– mice. The pancreatic duct expresses NHE1 in the basolateral membrane, and NHE2 and NHE3 in the luminal membrane, but does not contain NHE4 or NHE5. Basolateral Na⁺-dependent H⁺ efflux in the microperfused duct was inhibited by 1.5 μM of the amiloride analogue HOE 694, consistent with expression of NHE1, whereas the luminal activity required 50 μM HOE 694 for effective inhibition, suggesting that the efflux might be mediated by NHE2. However, disruption of NHE2 had no effect on luminal transport, while disruption of the NHE3 gene reduced luminal Na⁺-dependent H⁺ efflux by ∼45%. Notably, the remaining luminal Na⁺-dependent H⁺ efflux in ducts from NHE3^–/– mice was inhibited by 50 μM HOE 694. Hence, ∼55% of luminal H⁺ efflux (or HCO₃^– influx) in the pancreatic duct is mediated by a novel, HOE 694–sensitive, Na⁺-dependent mechanism. H⁺ transport by NHE3 and the novel transporter is inhibited by cAMP, albeit to different extents. We propose that multiple Na⁺-dependent mechanisms in the luminal membrane of the pancreatic duct absorb Na⁺ and HCO₃^– to produce a pancreatic juice that is poor in HCO₃^– and rich in Cl^– during basal secretion. Inhibition of the transporters during stimulated secretion aids in producing the HCO₃^–-rich pancreatic juice.