ClC-1 is the major chloride channel of adult skeletal muscle and necessary for electrical stability of muscle fibers. Dysfunction of human ClC-1 causes myotonia congenita, an inherited condition characterized by muscle stiffness upon sudden forceful movement. We here study the functional consequences of a disease-causing mutation, K231R, that was identified in a patient with recessive generalized myotonia (Becker). K231 is part of a highly conserved amino acid motif that contributes to the selectivity filter and forms a predicted activation gate of ClC channels. We transiently expressed WT and K231R hClC-1 channels in tsA201 cells and studied currents through whole-cell patch clamping. WT currents rise instantaneously upon voltage steps in the positive as well as in the negative direction. At positive potentials, current amplitudes are time-independent; upon membrane hyperpolarization the instantaneous rise is followed by a biexponential decrease of the current amplitude mediated by two kinetically distinct processes, i.e. fast and slow gating. WT channels assume low open probabilities at negative potentials and are activated by membrane depolarization. At the resting potential of muscle fibers, the absolute open probability is about 50%. K231R leaves fast gating virtually unaffected, but alters slow gating. K231R hClC-1 channels displays an extremely slow activation (t = 4.2 0.2 s at +105 mV) at positive potentials and a shift of the activation curve to more positive potentials. This shift causes a decreased percentage of open channels at the resting potential of skeletal muscle fibers (Popen < 0.1) and thus fully explains myotonia. Whereas activation of the slow gate of WT hClC-1 follows a simple Boltzmann distribution with maximum open probability at positive potentials, the slow gate activation curve of K231R hClC-1 is U-shaped with minimum open probability around -75 mV. Steps to negative and to positive voltages result in slow activation of K231R hClC-1. hClC-1 channels are known to be affected by external and internal pH, and we therefore studied gating of WT and K231R hClC-1 at various pH values. In K231R hClC-1, slow gate activation is promoted by alkalinisation at positive potentials and reduced at negative potentials. External acidification, however, makes slow activation upon hyperpolarization more pronounced and decreases slow activation upon depolarization. These findings suggest that the slow gate of K231R hClC-1 assumes two different protonation-dependent conformations with inverse voltage dependence.