hClC-1 chloride channels stabilize the resting membrane potential of skeletal muscle fibers. A monogenetic disease caused by mutations in this channel, myotonia congenita, is characterized by muscle stiffness as a result of an over-excitability of muscle fibers. We focused on four naturally occurring mutations found in patients with myotonia congenita, Q43R, S70L, Y137D and Q160H. We transiently expressed WT and mutant channels in tsA201 cells, and recorded whole-cell currents using standard patch-clamp techniques. Q43R, Y137D and Q160H, but not S70L, reduced macroscopic current amplitudes without major effects on the time and voltage dependence of ClC-1 currents. ClC-1 proteins are double-barreled channels with fast gating of the individual protopores and common slow gating that affects both protopores simultaneously. Time- and voltage-dependent gating of mutant channels remained comparable to WT channels even though minimum open probabilities of the slow gate were slightly increased for Q43R and Y137D. Noise analyses of macroscopic currents revealed that neither absolute open probabilities nor single channel conductances were affected by the mutations, indicating that the mutations exclusively affected subcellular distribution of ClC-1. We next investigated subcellular distribution of channels by confocal imaging using fluorescent fusion proteins of hClC-1 and mYFP expressed in MDCK II cells. While WT channels almost exclusively reside in the surface membrane, expression of mutant channels results in some intracellular staining of varying intensity. To quantify impaired intracellular trafficking we developed a novel assay using a combination of electrophysiological current recordings and whole-cell fluorescence measurements on the single cell level. With the exception of S70L, all tested mutations caused a significant reduction in the relative number of ClC-1 channels in the surface membrane explaining the disease causing effects of mutations Q43R, Y137D and Q160H. Heterodimeric channels expressed by transfection of concatameric WT-Q43R constructs have shown significantly less efficiency for inserting correctly into the membrane. In co-transfection experiments of WT YFP-hClC-1 and Q43R CFP-hClC-1 currents were reduced to a lower extent than predicted from fluorescence intensities under the assumption of binomially distributed homo- and heterodimeric channels. This suggests impaired interaction between WT and Q43R subunits. The new method offers a sensitive approach to investigate mutations that were reported to cause myotonia, but display only minor changes in electrophysiology.