Previous work has investigated the anion selectivity of transepithelial Cl- secretion by airway epithelia and its inhibition by the Cl(-)-channel blocker 5-nitro-2-(3-phenylpropylamino)benzoate (NPPB). Here we report the anion selectivity of the apical membrane and of the outwardly rectifying Cl- channel and the effect of NPPB on the Cl- channel. The anion selectivity sequence of the apical membrane determined with conventional microelectrodes in the native epithelium was SCN- greater than I- greater than Br- greater than NO3- approximately Cl- much greater than SO(4)2- approximately gluconate-. This contrasts with the observation that Cl- and Br- support transepithelial secretion but that I- does not. Thus the anion selectivity of transepithelial transport is determined by the basolateral membrane Cl- entry step. The anion selectivity of the outwardly rectifying Cl- channel studied in excised patches was the same as that of the apical membrane. We also found that NPPB reversibly blocked the outwardly rectifying Cl- channel from both the internal and external surfaces of the patch. NPPB, 10 microM, completely blocked the channel; lower concentrations caused a decrease in the probability of finding the channel in the open state. NPPB also caused the appearance of a subconductance state of the channel, an occurrence which is rarely observed in the absence of NPPB. These data provide further support for the conclusion that the outwardly rectifying Cl- channel is responsible for Cl- exit from the cell across the apical membrane.