Mutations in the voltage-gated chloride/proton antiporter ClC-5 gene, CLCN5, are associated with Dent's disease, an X-linked renal tubulopathy. Our interest is to identify and characterize disease-causing CLCN5 mutations, especially those that alter the splicing of the pre-mRNA. We analyzed the CLCN5 gene from nine unrelated Spanish Dent's disease patients and their relatives by DNA sequencing. Pre-mRNA splicing analysis was performed by RT-PCR. Seven new mutations were identified, consisting of three missense mutations (C219R, F273L, and W547G), one splice-site mutation (IVS-2A > G), one deletion (976delG), and two non-sense mutations (Y140X and W314X). We found that missense mutation W547G also led to increased expression of a new alternative isoform lacking exons 10 and 11 that was expressed in several human tissues. In addition, we describe another novel CLCN5 splicing variant lacking exon 11 alone, which was expressed only in human skeletal muscle. We conclude that missense mutation W547G can also alter the expression levels of a CLCN5 mRNA splicing variant. This type of mutation has not been previously described in the CLCN5 gene. Our results support the importance of a routine analysis at the pre-mRNA level of mutations that are commonly assumed to cause single amino acids alterations.