1. Three families with a form of myotonia (muscle stiffness due to membrane hyperexcitability) clinically distinct from previously classified myotonias were examined. The severity of the disease greatly differed among the families. 2. Three dominant point mutations were discovered at the same nucleotide position of the SCN4A gene encoding the adult skeletal muscle Na+ channel alpha-subunit. They predict the substitution of either glutamic acid, valine or alanine for glycine1306, a highly conserved residue within the supposed inactivation gate. Additional SCN4A mutations were excluded. 3. Electrophysiological studies were performed on biopsied muscle specimens obtained for each mutation. Patch clamp recordings on sarcolemmal blebs revealed an increase in the time constant of fast Na+ channel inactivation, tau h, and in late channel openings as compared to normal controls. tau h was increased from 1.2 to 1.6-2.1 ms and the average late currents from 0.4 to 1-6% of the peak early current. 4. Intracellular recordings on resealed fibre segments revealed an abnormal tetrodotoxin-sensitive steady-state inward current, and repetitive action potentials. Since K+ and Cl- conductances were normal, only the increase in the number of non-inactivating Na+ channels has to be responsible for the membrane hyperexcitability. 5. Length, ramification and charge of the side-chains of the substitutions correlated well with the Na+ channel dysfunction and the severity of myotonia, with alanine as the most benign and glutamic acid as the substitution with a major steric effect. 6. Our electrophysiological and molecular genetic studies strongly suggest that these Na+ channel mutations cause myotonia. The naturally occurring mutants allowed us to gain further insight into the mechanism of Na+ channel inactivation.