Lyotropic pseudohalide anions are potentially useful as high affinity probes of Cl(-) channel pores. However, the interaction between these pseudohalides and the cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channel have not been described in detail. Here we show that Au(CN)(2-) ions applied to the intracellular face of membrane patches from stably transfected baby hamster kidney cells inhibit CFTR channel currents by at least two mechanisms, which can be distinguished at the single channel level or by inhibiting channel closure using 2 mM pyrophosphate. Low concentrations (< 10 microM) of Au(CN)(2-) significantly reduced CFTR channel open probability. This effect was apparently voltage insensitive, independent of extracellular Cl(-) concentration, and lost following exposure to pyrophosphate. Higher concentrations of intracellular Au(CN)(2-) caused an apparent reduction in unitary current amplitude, presumably due to a kinetically fast blocking reaction. This effect, isolated following exposure to pyrophosphate, was strongly voltage dependent (apparent K(d) 61.6 microM at -100 mV and 913 microM at +60 mV). Both the affinity and voltage dependence of block were highly sensitive to extracellular Cl(-) concentration. We propose that Au(CN)(2-) has at least two inhibitory effects on CFTR currents: a high affinity effect on channel gating due to action on a cytoplasmically accessible aspect of the channel and a lower affinity block within the open channel pore. These results offer important caveats for the use of lyotropic pseudohalide anions such as Au(CN)(2-) as specific high affinity probes of Cl(-) channel pores.