Mutations of the major myelin gene, proteolipid protein (Plp), cause Pelizaeus-Merzbacher disease and some forms of spastic paraplegia in man and dysmyelinating phenotypes in animals. The clinical severity is markedly heterogeneous, ranging from relatively mild to severe and fatal. Point mutations, or frame shifts, which are predicted to result in translation of structurally altered proteins account for many of these cases, including 3 of the allelic murine conditions. Plp(jp-rsh), Plp(jp-msd), and Plp(jp) represent an increasing severity of clinical and pathological phenotypes, respectively. In this study we determined whether there was any correlation between the severity of phenotype and the transport of the predicted abnormal protein. We examined the ability of the two products of the Plp gene, PLP and DM20, to insert into the plasma membrane of transfected BHK or COS-7 cells, and into the myelin sheath of oligodendrocytes. With these complementary in vitro and in vivo approaches we find that proteins of Plp(jp-rsh), associated with the mildest phenotype, have a far greater ability to insert into the cell membrane or myelin than those associated with the severe phenotypes. Additionally, altered DM20 is more readily transported to the cell surface and to myelin than the PLP isoprotein. Interestingly, the two clonal cell lines chosen for transient transfection differ in their ability to fold DM20 from Plp(jp-rsh) and Plp(jp-msd) mice correctly, as inferred by staining for the conformation-sensitive O10 epitope. In the case of Plp(jp), which is associated with the most severe phenotype, no PLP or O10 staining is present at the cell surface or in myelin. The perturbation in trafficking observed for altered Plp(jp) PLP and DM20 in oligodendrocytes does not extend to other myelin membrane proteins, such as MAG and MOG, nor to wild type PLP co-expressed in the same cell, all of which are correctly inserted into myelin. As Plp-knockout mice do not have a dysmyelinating phenotype it seems unlikely that absence of PLP and/or DM20 in the membrane is responsible for the pathology. It remains to be determined whether the perturbation in protein trafficking is associated with the dysmyelination, or if the altered product of the mutant alleles acquire a novel function which is deleterious to myelin production by oligodendrocytes.