Corneal transparency and hydration control are dependent on the HCO₃^- transport properties of the corneal endothelium. A recent study (13) suggests the presence of an apical 1Na⁺/3HCO₃^- cotransporter (NBC1) in addition to a basolateral 1Na⁺/2HCO₃^- cotransporter. Here, we ask if the Sodium Bicarbonate Cotransporter (NBC-1) contributes significantly to basolateral or apical HCO₃^- permeability and whether the cotransporter participates in transendothelial net HCO₃^- flux in cultured bovine corneal endothelium. NBC1 protein expression was reduced by using siRNA. Immunoblot analysis showed that 5-15 nM siRNA decreased NBC1 expression by 80-95%, 4 days post-transfection. Apical and basolateral HCO₃^- permeabilities were determined by measuring the rate of pHi change when HCO₃^- was removed from the bath under Constant pH or Constant CO₂ conditions. Using either protocol we found that cultures treated with NBC1 siRNA had 6-fold lower basolateral HCO₃^- permeability compared to untreated or siCONTROL siRNA treated cells. Apical HCO₃^- permeability was unaffected by NBC1 siRNA treatment. Net non-steady-state HCO₃^- flux was 0.707 ± 0.009 mM/min x cm² in the basolateral (B) to apical (A) direction and was increased to 1.74±0.15 when cells were stimulated with 2 µM forskolin. Treatment with 5 nM siRNA decreased B to A flux by 67%, while A to B flux was unaffected, significantly decreasing net HCO₃^- flux to 0.236±0.002. NBC1 siRNA treatment or 100 µM ouabain also eliminated steady-state HCO₃^- flux, as measured by apical compartment alkalinization. Collectively, reduced basolateral HCO₃^- permeability, B to A fluxes, and net HCO₃^- flux as a result of reduced expression of NBC1 indicates that NBC1 plays a key role in transendothelial HCO₃^- flux and is functional only at the basolateral membrane.