Nephropathic cystinosis is an autosomal recessive lysosomal storage disease characterized by renal failure at 10 years of age and other systemic complications. The gene for cystinosis, CTNS, has 12 exons. Its 2.6-kb mRNA codes for a 367-amino-acid putative cystine transporter with seven transmembrane domains. Previously reported mutations include a 65-kb 'European' deletion involving marker D17S829 and 11 small mutations. Mutation analysis of 108 American-based nephropathic cystinosis patients revealed that 48 patients (44%) were homozygous for the 65-kb deletion, 2 had a smaller major deletion, 11 were homozygous and 3 were heterozygous for 753G→A (W138X), and 24 had 21 other mutations. In 20 patients (19%), no mutations were found. Of 82 alleles bearing the 65-kb deletion, 38 derived from Germany, 28 from the British Isles, and 4 from Iceland. Eighteen new mutations were identified, including the first reported missense mutations, two inframe deletions, and mutations in patients of African American, Mexican, and Indian ancestry. CTNS mutations are spread throughout the leader sequence, transmembrane, and nontransmembrane regions. According to a cystinosis clinical severity score, homozygotes for the 65-kb deletion and for W138X have average disease, whereas mutations involving the first amino acids prior to transmembrane domains are associated with mild disease. By northern blot analysis, CTNS was not expressed in patients homozygous for the 65-kb deletion but was expressed in all 15 other patients tested. These data demonstrate the origins of CTNS mutations in America and provide a basis for possible molecular diagnosis in this population.