Molecular genetic studies have shown that the peripheral myelin protein 22 (PMP22) is a key gene in hereditary peripheral neuropathies and appears to be essential for the formation and maintenance of myelin in the PNS. Based on the amino acid sequence the predicted structure of PMP22 protein contains two major distinct hydrophilic regions and four transmembrane domains. To analyze the cellular localization and membrane topology of PMP22 we inserted an octapeptide tag-sequence at the amino or at the carboxyl terminus of the PMP22 open reading frame and generated different chimeric constructs which were expressed in HeLa cells. The expression of the tagged PMP22 protein and its orientation with respect to the plasma membrane were analyzed using antibodies raised against specific PMP22 epitopes and the tag sequence. Combined indirect, double-immunofluorescence labeling and confocal microscopy showed that PMP22 is synthesized in the rough endoplasmic reticulum of transfected cells and passes through the Golgi apparatus to the cell surface. We determined the transmembrane organization of PMP22 providing the first experimental evidence that confirms the cytoplasmic disposition of its N and C termini and the extracellular localization of the two hydrophilic domains containing amino acids 28-40 and 118-131. This study provides the basis for further analysis aimed to identify functional domains of wild-type PMP22 and the cellular sorting of mutant forms of PMP22.