SLC26 anion exchangers (probably SLC26A3 and SLC26A6) are expressed on the apical membrane of pancreatic duct cells and play a key role in HCO 3- secretion; a process that is inhibited by the neuropeptide, substance P (SP). SP had no effect on basolateral HCO 3- transporters in the duct cell or on CFTR Cl- channels, but inhibited a Cl--dependent HCO3- efflux step on the apical membrane. In microperfused ducts, luminal H 2DIDS (0.5 mM) caused intracellular pH to alkalinize (consistent with inhibition of HCO3- efflux) and, like SP, inhibited HCO3- secretion. SP did not reduce HCO3 - secretion further when H2DIDS was applied to the duct lumen, suggesting that SP and H2DIDS inhibit the same transporter on the apical membrane. As SLC26A6 is DIDS-sensitive, this isoform is the likely target for SP. The inhibitory effect of SP was mimicked by phorbol 12,13-dibutyrate (PDBu), an activator of protein kinase C (PKC). Moreover, bisindolylmaleimide, a blocker of PKC, relieved the inhibitory effect of both SP and PDBu on HCO3- secretion. Western blot analysis revealed that guinea pig pancreatic ducts express the α, β1, δ, ε, η, θ, ζ and μ isoforms of PKC. We conclude that PKC is a negative regulator of SLC26 activity in pancreatic duct cells.