Quaternary ammonium (QA) compounds inhibit K+ conductance by entering and occluding the open pore of voltage-activated K+ channels. We characterized the effects of a series of alkyl-triethylammonium blockers on the Shaker K+ channel and tested them on a series of site-directed mutants of the channel protein in order to define the structural features of the binding sites. We found that mutations in two regions of the channel protein, the pore (P) region and the last transmembrane sequence (S6), appear to alter QA binding, not through their effects on gating but perhaps through direct effects on the binding site. Several mutations in the P region affect tetraethylammonium binding but have minimal effects on longer blockers, suggesting that the hydrophobic tail contributes to binding in a nonadditive fashion. Binding of the longer blockers can be affected by varying the hydrophobicity of 1 residue within S6 by site-specific substitution, in a manner consistent with a direct hydrophobic interaction between the side chain at this site and the alkyl chains of the blocker.