Niemann-Pick disease type C (NPC), a neurovisceral disorder characterized by accumulation of cholesterol and glycolipids in the lysosomal/late endosomal system, is due to mutations on either the NPC1 or the NPC2 genes. Although NPC1 and NPC2 proteins appear essential for proper cellular cholesterol trafficking, their precise functions and relationship have remained elusive. Mutation identification in NPC2 patients did not provide insights into structure-function relationships, but recent studies brought important information on the cholesterol-binding site of the NPC2 protein. The present work was focused on localization and N-glycosylation of NPC2, considering that glycosylation is often essential for targeting, stability and biological function of proteins. Using immunocytofluorescence in cultured human fibroblasts, we found that the native NPC2 protein is essentially lysosomal, at variance with the late endosomal location of NPC1. Expression of cDNA mutants affecting each of the three potential NPC2 N-glycosylation sites in NPC2-/- fibroblasts showed that only two sites are used. The intracellular human NPC2 protein occurred as two N-glycosylated forms, with either one single oligosaccharide chain attached to Asn 58 or two oligosaccharides attached to Asn 58 and 135. The oligosaccharidic chains were of the hybrid and/or high mannose type, with no complex chains. Further studies on the cellular location of Asn 58 and Asn 135 mutant proteins and their respective effect on restoration of normal cholesterol traficking in NPC2-/- cells led to the conclusion that only the oligosaccharide chain carried by Asn 58 is responsible for proper targeting of NPC2 to lysosomes, and is crucial for NPC2 function.