Batrachotoxin (BTX) is a steroidal alkaloid that causes Na+ channels to open persistently. This toxin has been used widely as a tool for studying Na+ channel gating processes and for estimating Na+ channel density. In this report we used point mutations to identify critical residues involved in BTX binding and to examine if such mutations affect channel gating. We show that a single asparagine --> lysine substitution of the rat muscle Na+ channel alpha-subunit, mu1-N434K, renders the channel completely insensitive to 5 microM BTX when expressed in mammalian cells. This mutant channel nonetheless displays normal current kinetics with minimal changes in gating properties. Another substitution, mu1-N434A, yields a partial BTX-sensitive mutant. Unlike wild-type currents, the BTX-modified mu1-N434A currents continue to undergo fast and slow inactivation as if the inactivation processes remain functional. This finding implies that the mu1-N434 residue upon binding with BTX is critical for subsequent changes on gating; alanine at the mu1-434 position apparently diminishes the efficacy of BTX on eliminating Na+ channel inactivation. Mutants of two adjacent residues, mu1-I433K and mu1-L437K, also were found to exhibit the identical BTX-resistant phenotype. We propose that the mu1-I433, mu1-N434, and mu1-L437 residues in transmembrane segment I-S6 probably form a part of the BTX receptor.