To study in detail the roles of basic amino acid residues in the activity of μ‐conotoxin GIIIA (μ‐GIIIA) and GIIIB (μ‐GIIIB), specific blockers of muscle sodium channels, seven analogs of μ‐GIIIA, and two analogs of μ‐GIIIB were synthesized. μ‐GIIIA analogs were synthesized by replacing systematically the three Arg residues (Arg1, Arg13, and Arg19) with one, two, and three Lys residues. μ‐GIIIB analogs were synthesized by replacing simultaneously all four Lys residues (Lys9, Lys11, Lys16, and Lys19) with Arg residues and further replacement of acidic Asp residues with neutral Ala residues. Circular dichroism spectra of the synthesized analogs suggested that the replacement did not affect the three dimensional structure. The inhibitory effects on the twitch contractions of the rat diaphragm showed that the side chain guanidino group of Arg13 of μ‐GIIIA was important for the activity, whereas that of Arg19 had little role for biological activity. Although [Arg9,11,16,19]μ‐GIIIB showed higher activity than native μ‐GIIIB, highly basic [Ala2,12, Arg9,11,16,19]μ‐GIIIB showed lower activity, suggesting that there was an appropriate molecular basicity for the maximum activity.