The muscle chloride channel CIC-1 regulates the electric excitability of the skeletal muscle membrane. Mutations in the gene encoding this chloride channel (CLCN1) are responsible for both human purely myotonic disorders, autosomal recessive generalized myotonia (Becker's disease, GM) and autosomal dominant myotonia congenita (Thomsen's disease, MC). We now show that the protein-coding sequence of the CLCN1 gene is organized into 23 exons. The CIC-1 upstream region contains a canonical TATA box, several consensus binding sites for myogenic transcription factors and two other putative regulatory elements. SSCA analysis of a German GM family revealed that affected members are compound heterozygotes having two novel mutations. G979A affects a splice consensus site at the end of exon 8, and G1488T in exon 14 leads to a replacement of a positive charge in a highly conserved putative transmembrane domain (R496S). Functional expression of R496S cRNA in Xenopus oocytes did not yield detectable currents. It neither suppressed wild-type currents in a co-expression assay, confirming it as a recessive mutation.