Voltage-gated sodium channels are important in initiating and propagating nerve impulses in various tissues, including cardiac muscle, skeletal muscle, the brain, and the peripheral nerves. Hyperexcitability of these channels leads to such disorders as cardiac arrhythmias (Na(v)1.5), myotonias (Na(v)1.4), epilepsies (Na(v)1.2), and pain (Na(v)1.7). Thus, there is strong motivation to identify isoform-specific blockers and the molecular determinants underlying their selectivity among these channels. μ-Conotoxin KIIIA blocks rNa(v)1.2 (IC(50), 5 nM), rNa(v)1.4 (37 nM), and hNa(v)1.7 (97 nM), expressed in mammalian cells, with high affinity and a maximal block at saturating concentrations of 90 to 95%. Mutations of charged residues on both the toxin and channel modulate the maximal block and/or affinity of KIIIA. Two toxin substitutions, K7A and R10A, modulate the maximal block (52-70%). KIIIA-H12A and R14A were the only derivatives tested that altered Na(v) isoform specificity. KIIIA-R14A showed the highest affinity for Na(v)1.7, a channel involved in pain signaling. Wild-type KIIIA has a 2-fold higher affinity for Na(v)1.4 than for Na(v)1.7, which can be attributed to a missing outer vestibule charge in domain III of Na(v)1.7. Reciprocal mutations Na(v)1.4 D1241I and Na(v)1.7 I1410D remove the affinity differences between these two channels for wild-type KIIIA without affecting their affinities for KIIIA-R14A. KIIIA is the first μ-conotoxin to show enhanced activity as pH is lowered, apparently resulting from titration of the free N terminus. Removal of this free amino group reduced the pH sensitivity by 10-fold. Recognition of these molecular determinants of KIIIA block may facilitate further development of subtype-specific, sodium channel blockers to treat hyperexcitability disorders.