Missense mutations in the skeletal muscle sodium channel alpha-subunit gene (SCN4A) are associated with a group of clinically overlapping diseases caused by alterations in the excitability of the sarcolemma. Sodium channel defects may increase excitability and cause myotonic stiffness or may render fibres transiently inexcitable to produce periodic paralysis. A patient with cold-aggravated myotonia did not harbour any of the common SCN4A mutations. We therefore screened all 24 exons by denaturing high-performance liquid chromatography, followed by direct sequencing. Two novel missense changes were found with predicted amino acid substitutions: T323M in the DIS5-S6 loop and F1705I in the intracellular C-terminus. The functional impact of these substitutions was assessed by recording whole-cell Na+ currents from transiently transfected HEK293 cells. T323M currents were indistinguishable from wild-type (WT). Fast inactivation was impaired for F1705I channels, as demonstrated by an 8.6-mV rightwards shift in voltage dependence and a two-fold slowing in the rate of inactivation. Recovery from fast inactivation was not altered, nor was there an increase in the persistent current after a 50- ms depolarization. Activation and slow inactivation were not appreciably affected. These data suggest that T323M is a benign polymorphism, whereas F1705I results in fast inactivation defects, which are often observed for myotonia. This is the first example of a C-terminal mutation in SCN4A associated with human disease. Like the cardiac disorders (long QT syndrome type 3 or Brugada syndrome) and generalized epilepsy with febrile seizures plus (GEFS+) associated with C-terminal mutations in other NaV channels, the primary effect of F1705I was a partial disruption of fast inactivation.