The Na + channel α-subunit contains an IFM motif that is critical for the fast inactivation process. In this study, we sought to determine whether an IFM-containing peptide, acetyl-KIFMK-amide, blocks open cardiac Na + channels via the inner cavity. Intracellular acetyl-KIFMK-amide at 2mM elicited a rapid time-dependent block (τ=0.24ms) of inactivation-deficient human heart Na + channels (hNav1.5-L409C/A410W) at +50mV. In addition, a peptide-induced tail current appeared conspicuously upon repolarization, suggesting that the activation gate cannot close until acetyl-KIFMK-amide is cleared from the open pore. Repetitive pulses (+50mV for 20ms at 1Hz) produced a substantial use-dependent block of both peak and tail currents by ∼65%. A F1760K mutation (hNav1.5-L409C/A410W/F1760K) abolished the use-dependent block by acetyl-KIFMK-amide and hindered the time-dependent block. Competition experiments showed that acetyl-KIFMK-amide antagonized bupivacaine binding. These results are consistent with a model that two acetyl-KIFMK-amide receptors exist in proximity within the Na + channel inner cavity.