Anion selectivity of the cystic fibrosis conductance transmembrane conductance regulator (CFTR) and other channels and parallel pathways expressed endogenously in apical membranes of polarized Calu-3 epithelial monolayers was studied under control conditions and during cAMP stimulation. Basolateral membranes were eliminated using alpha-toxin. The cAMP-stimulated, gradient-driven currents had the sequence Br≥Cl≥NO₃>SCN> I≥F>formate>HCO₃>acetate>propionate=butyrate=ATP= PPi=PO₄=SO₄=0. Selectivity of parallel cAMP-independent pathway(s) was Br>Cl=SCN=NO₃>I>formate=F >HCO₃>acetate>propionate. SCN, I, F or formate blocked cAMP-stimulated, but not control, Cl currents. Anions >0.53 nm in diameter were impermeant, suggesting that the apical CFTR channel has a limiting diameter of 0.53 nm. The selectivity, blocking patterns and pore size of the cAMP-stimulated conductance pathway were very similar to those in previous reports in which CFTR was heterologously expressed in non-epithelial cells. Thus, CFTR appears to be the major apical anion conductance pathway in Calu-3 cells, and its conduction properties are independent of the expression system. CFTR in Calu-3 cells also conducts physiologically relevant anions, but not ATP, PO₄ or SO₄. A pathway parallel (probably a tight junction) showed a different selectivity than CFTR.