The cystic fibrosis transmembrane conductance regulator (CFTR) contributes to transport of Cl- and HCO3- across epithelial apical membranes, and epithelial secretion of both of these anions is defective in cystic fibrosis. The extracellular face of CFTR is exposed to widely varying concentrations of Cl- and HCO3- in secreted fluid, most strikingly in the pancreas but also in other epithelial tissues. Here we present evidence that extracellular HCO3- regulates CFTR conductance by a novel mechanism that involves trans-channel interactions with intracellular blocking anions. It has long been supposed that CFTR currents in intact cells are susceptible to voltage-dependent block by unknown cytosolic anions (1), leading to outward rectification of the current-voltage relationship. However, the molecular mechanism or physiological relevance of block are not known. We used cell-attached and inside-out patch clamp recording to monitor macroscopic currents carried by constitutively active mutant E1371Q-CFTR channels transiently expressed in BHK cells. As described recently (2), comparing currents in cell-attached patches with those immediately following patch excision allowed a simple method to quantify the strength of channel block in intact cells. The strength of block quantified in this way was significantly (p<0.02) reduced by mutations in the pore that are known to weaken interactions with open channel blockers (K95Q, R303Q, K978Q). Interestingly, the strength of block in intact cells was significantly increased when extracellular Cl- (154 mM) was partly reduced by equimolar replacement by HCO3- (50-150 mM; p<0.05). This appears to reflect differences in the ability of extracellular Cl- and HCO3- ions to interact with and repel intracellular blocking ions from the pore, since open channel block by internal 5-nitro-2-(3-phenylpropylamino)benzoic acid in inside-out patches was also significantly stronger when external Cl- was replaced by HCO3- (p<0.05). Strong apparent block by cytosolic anions in intact cells with HCO3–containing solutions occurs predominantly at physiologically relevant hyperpolarised membrane potentials and can result in up to ~50% inhibition of current amplitude compared to Cl–containing solutions. We propose that channel block by cytosolic anions is a physiologically relevant mechanism of channel regulation that confers CFTR channels with sensitivity to different anions in the extracellular fluid. Our results suggest that CFTR-dependent anion transport will be maximized during periods of epithelial Cl- secretion, and minimized when the epithelium secretes HCO3–rich fluid. External anion sensitivity therefore represents a feedback mechanism by which CFTR-dependent anion secretion could be regulated by the composition of the secretions themselves.