The extracellular side of single batrachotoxin-activated voltage-dependent Na channels isolated from rat skeletal muscle membranes incorporated into neutral planar lipid bilayers were treated in situ with the carboxyl methylating reagent, trimethyloxonium (TMO). These experiments were designed to determine whether TMO alters Na channel function by a general through-space electrostatic mechanism or by methylating specific carboxyl groups essential to channel function. TMO modification reduced single-channel conductance by decreasing the maximal turnover rate. Modification increased channel selectivity for sodium ions relative to potassium ions as measured under biionic conditions. TMO modification increased the mu-conotoxin (muCTX) off-rate by three orders of magnitude. Modification did not alter the muCTX on-rate at low ionic strength or Na channel voltage-dependent gating characteristics. These data demonstrate that TMO does not act via a general electrostatic mechanism. Instead, TMO targets protein residues specifically involved in ion conduction, ion selectivity, and muCTX binding. These data support the hypothesis that muCTX blocks open-channel current by physically obstructing the ion channel pore.