Voltage-gated ClC-1 chloride channels encoded by the CLCN1 gene have a major role in setting the membrane potential in skeletal muscle. More than 60 CLCN1 mutations have been associated with myotonia congenita. These mutations are traditionally classified as recessive (Becker's disease) or dominant (Thomsen's disease). In this study, we have electrophysiologically characterized two new dominant ClC-1 mutations, thereby elucidating the observed phenotype in patients. The two ClC-1 mutants M128V and E193K were identified, and the DNA was isolated from patients and subsequently expressed in Xenopus laevis oocytes for electrophysiological characterization. Both ClC-1 mutants, M128V and E193K, showed a large rightward shift in the current-voltage relationship. In addition, the activation kinetics were slowed in the ClC-1 M128V mutant, as compared to the wild-type ClC-1. Interestingly, ClC-1 E193K revealed a change in reversal potential compared to wild-type channels. This finding supports the notion that the E193 amino acid is an important determinant in the selectivity filter of the human ClC-1 channel. The electrophysiological behavior of both mutants demonstrates a severe reduction in ClC-1 channel conductance under physiologically relevant membrane potentials. These studies thereby explain the molecular background for the observed myotonia in patients.