Human NBC3 is an electroneutral Na⁺/HCO₃^- co-transporter expressed in heart, skeletal muscle, and kidney where it plays an important role in HCO₃^- metabolism. Cytosolic enzyme, Carbonic Anhydrase II (CAII) catalyzes the reaction: CO₂+H₂O HCO₃^-+H⁺ in many tissues. We investigated whether NBC3, like some Cl^-/HCO₃^- exchange proteins, could bind CAII and whether protein kinase A (PKA) could regulate NBC3 activity through modulation of CAII binding. CAII bound the C-terminal domain of NBC3 (NBC3Ct) with Kd=101 nM; the interaction was stronger at acid pH. Co-transfection of HEK293 cells with NBC3 and CAII recruited CAII to the plasma membrane. Mutagenesis of consensus CAII binding sites revealed that the D1135-D1136 region of NBC3 is essential for CAII/NBC3 interaction and for function since the NBC3 D1135N/D1136N retained only 29% ± 22% of wild-type activity. Co-expression of the functionally dominant negative CAII mutant V143Y with NBC3 or addition of 100 mM 8-Br-cAMP to NBC3 transfected cells reduced pHi recovery rate by 31% ± 3%, or 38% ± 7%, respectively, relative to untreated NBC3 transfected cells. The effects were additive, together decreasing pHi recovery rate by 69% ± 12%, suggesting that PKA reduces transport activity by a mechanism independent of CAII. Measurements of PKA-dependent phosphorylation by mass spectroscopy and labelling with -³²P-ATP, showed that NBC3Ct was not a PKA substrate. These results demonstrate that NBC3 and CAII interact to maximize the HCO₃^- transport rate. While PKA decreased NBC3 transport activity, it did so independent of the NBC3/CAII interaction, and did not involve phosphorylation of NBC3Ct.