The cytoplasmic domain of myelin protein zero (MPZ), the principal protein of peripheral myelin, undergoes phosphorylation on several serine residues and a tyrosine group that is maximal during peak nerve myelination. Mutations that could affect MPZ phosphorylation cause the inherited neuropathy, Charcot-Marie-Tooth disease Type 1B. To investigate a possible role for phosphorylation in regulation of MPZ trafficking within the cell, we expressed wild-type and mutated MPZ-enhanced green fluorescent protein (GFP) fusion proteins in cultured Schwann-like cells. Whereas wild-type protein is present almost entirely at the cell surface, mutation of serine 204 to alanine or at a nearby presumed PKC substrate motif (198RSTK201) causes 40–60% of protein to be retained in the cytoplasm. Mutation of S204 to aspartate, which introduces a permanent negative charge, also impairs MPZ movement to the plasma membrane. In contrast, tyrosine 191 mutation has no effect on MPZ cellular distribution. Simultaneous alteration of S204 and Y191 produces much less perturbation of MPZ trafficking than mutation of S204 alone. Colocalization studies showed that mutated MPZ-EGFP trapped in the cytoplasm associates with all organelles in the secretory pathway. Previous studies have shown that cytoplasmic mutations at serine, but not tyrosine phosphorylation sites, abolish MPZ adhesive properties. Our results suggest that this loss of adhesion may be due, at least in part, to a failure of sufficient MPZ to reach the cell surface and that this impaired trafficking is associated with deficient serine phosphorylation in the cytoplasmic domain.