cAMP-dependent activation of the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) regulates fluid transport in many tissues. Fluid secretion by the corneal endothelium is stimulated by cAMP and is dependent on the presence of HCO₃^-, which contributes to transendothelial anion transport. In this study, we ask if HCO₃^- can secondarily increase the permeability of apical CFTR in bovine corneal endothelial cells (BCEC) by activating Soluble Adenylyl Cyclase (sAC), which has defined a novel means for generating cAMP. Immunofluorescence of cultured BCEC suggests that sAC is distributed throughout the cytoplasm. The presence of 40 mM HCO₃^- increased total cellular [cAMP] 42% in the presence of 50 µM Rolipram (a PDE4 inhibitor) and a standard HCO₃^- Ringer's solution (28.5 mM) increased apical Cl^- permeability by 78% relative to HCO₃^--free Ringer's. The HCO₃^--dependent increase in Cl^- permeability was reduced 60% by 20 mM NaHSO₃^- (a weak agonist of sAC). NaHSO₃^- alone in HCO₃^--free Ringer's increased apical Cl^- permeability by only 13%. The HCO₃^--dependent increase in Cl^- permeability was reduced 57% in the presence of 50 mM Rp-cAMPS, and 86% by 50 mM NPPB applied to the apical side, but unaffected by 200 mM apical H₂DIDS. Phosphorylation of CFTR was increased 23%, 150%, and 32% by 20 mM HSO₃^-, 28.5 mM HCO₃^-, and 28.5 mM HCO₃^- + 20 mM HSO₃^-, respectively. Activation of apical Cl^- permeability by 5 µM genistein, a signature property of CFTR, was increased synergistically by HCO₃^- over that due to genistein and HCO₃^-- alone. We conclude that HCO₃^--stimulated sAC represents a form of autocrine signaling that contributes to baseline cAMP production thereby affecting baseline CFTR activity in BCEC. In general, this form of autocrine signaling may be particularly important in tissues that express sAC and exhibit robust HCO₃^-- influx (e.g., ciliary epithelium of the eye, choroid plexus, and airway epithelium).