Tetraethylammonium (TEA) has been used recently to probe natural and mutational variants of voltage-dependent K+ channels encoded by cDNA clones. Its usefulness as a probe of channel structure prompted us to examine the molecular mechanism by which TEA blocks single-channel currents in Xenopus oocytes expressing the rat brain K+ channel, RCK2. TEA at the intracellular surface of membrane patches decreased channel open time and increased the duration of closed intervals. Tetrapentylammonium had similar but more potent effects. Extracellular application of TEA caused an apparent reduction of single-channel amplitude. Block was slower at the high-affinity internal site than at the low-affinity external site. Internal TEA selectively blocks open K+ channels, and the voltage dependence of the block indicates that the binding site lies within the membrane electric field at a point 25% of the distance from the cytoplasmic margin. External TEA also interacts with the open channel but is less sensitive to membrane potential. The results indicate that the internal and external TEA binding sites define the inner and outer margins of the aqueous pore.