The III-IV linker (L(III-IV)) of the rat brain sodium channel is critical for fast inactivation, possibly forming a fast inactivation particle. Inactivation can be disrupted by mutation of a conserved alanine at position 1329 in the S4-S5 loop of domain III. Combination of a charged mutation at 1329 with a compensatory (opposite) charge mutation at position 1489 in L(III-IV) partially restores inactivation of the channel. The compensatory charge mutant channel has a single-channel mean open time that is similar to that of the wild-type channel and is approximately 50 times shorter than that of the L(III-IV) mutant channel. The results of thermodynamic cycle analysis indicate that the mutations in domain III S4-S5 and L(III-IV) have a coupling energy of 2.8 kcal/mol, indicating that the two mutations act interdependently. These data suggest that L(III-IV) interacts directly with A1329, which may form part of the docking site if L(III-IV) is a fast inactivation particle.