Nephropathic cystinosis is a lysosomal disorder caused by functional defects of cystinosin, which mediates cystine efflux into the cytosol. The protein sequence contains at least two signals that target the protein to the lysosomal compartment, one of which is located at the carboxy-terminal tail (GYDQL). We have isolated from a human kidney cDNA library a cystinosin isoform, which is generated by an alternative splicing of exon 12 that removes the GYDQL motif. Based on its last 3 amino acids, we have termed this protein cystinosin-LKG. Contrary to the lysosomal cystinosin isoform, expression experiments performed by transient transfection of GFP-fusion plasmids in HK2 cells, showed that cystinosin-LKG is expressed in the plasma membrane, in lysosomes and in other cytosolic structures. This sub-cellular localization of the protein was confirmed by transmission electron microscopy. In addition, immunogold labeling was observed in the endoplasmic reticulum and in the Golgi apparatus. Expression of the protein in renal tubular structures was also directly demonstrated by immunostaining of normal human kidney sections. The plasma membrane localization of cystinosin-LKG was directly tested by ³⁵S-cystine flux experiments in COS-1 cells. In the presence of a proton gradient, a marked enhancement of intracellular cystine transport was observed in cells over-expressing this isoform. These data indicate that the expression of the gene products encoded by the CTNS gene are not restricted to the lysosomal compartment. These finding may help elucidating the mechanisms of cell dysfunction in this disorder.