Nephropathic cystinosis is an autosomal recessive disorder caused by the defective transport of cystine out of lysosomes. Recently, the causative gene (CTNS) was identified and presumed to encode an integral membrane protein called cystinosin. Many of the disease-associated mutations inCTNSare deletions, including one >55 kb in size that represents the most common cystinosis allele encountered to date. In an effort to determine the precise genomic organization ofCTNSand to gain sequence-based insight about the DNA within and flanking cystinosis-associated deletions, we mapped and sequenced the region of human chromosome 17p13 encompassingCTNS. Specifically, a bacterial artificial chromosome (BAC)-based physical map spanningCTNSwas constructed by sequence-tagged site (STS)-content mapping. The resulting BAC contig provided the relative order of 43 STSs. Two overlapping BACs, which together contain all of theCTNSexons as well as extensive amounts of flanking DNA, were selected and subjected to shotgun sequencing. A total of 200,237 bp of contiguous, high-accuracy sequence was generated. Analysis of the resulting data revealed a number of interesting features about this genomic region, including the long-range organization ofCTNS, insight about the breakpoints and intervening DNA associated with the common cystinosis-causing deletion, and structural information about five genes neighboringCTNS(human ortholog of rat vanilloid receptor subtype 1 gene,CARKL, TIP-1, P2X5, andHUMINAE). In particular, sequence analysis detected the presence of a novel gene (CARKL) residing within the most common cystinosis-causing deletion. This gene encodes a previously unknown protein that is predicted to function as a carbohydrate kinase. Interestingly, bothCTNSandCARKLare absent in nearly half of all cystinosis patients (i.e., those homozygous for the common deletion).[The sequence data described in this paper have been submitted to the GenBank data library under accession nos.AF168787andAF163573.]