Open-channel blockers such as tetraethylammonium (TEA) have a long history as probes of the permeation pathway of ion channels.High affinity blockade by extracellular TEA requires the presence of an aromatic amino acid at a position that sits at the external entrance of the permeation pathway (residue 449 in the eukaryotic voltage-gated potassium channel Shaker).We investigated whether a cation-π interaction between TEA and such an aromatic residue contributes to TEA block using the in vivo nonsense suppression method to incorporate a series of increasingly fluorinated phenylalanine (Phe) side chains at position 449.Fluorination, which is known to decrease the cation-π binding ability of an aromatic ring, progressively increased the inhibitory constant Ki for the TEA block of Shaker.A larger increase in Ki was observed when the benzene ring of Phe449 was substituted by nonaromatic cyclohexane.These results support a strong cation-π component to the TEA block.The data provide an empirical basis for choosing between Shaker models that are based on two classes of reported crystal structures for the bacterial channel KcsA, showing residue tyrosine 82 (Tyr82) in orientations either compatible or incompatible with a cation-π mechanism.We propose that the aromatic residue at this position in Shaker is favorably oriented for a cation-π interaction with the permeation pathway.This choice is supported by high level ab initio calculations of the predicted effects of Phe modifications on TEA binding energy.