The topology of the integral membrane proteolipid protein (PLP) has important structural and functional implications for central nervous system myelin. To determine the orientation of the carboxyl-terminal portion of PLP, cultured mouse oligodendrocytes were probed with polyclonal antibodies raised against a synthetic terminal peptide corresponding to PLP residues 264-276 and with ten separate monoclonal antibodies that react with this region. Cells were examined by double-label indirect immunofluorescence for the presence of the PLP C-terminus and either oligodendrocyte-specific surface or intracellular antigens. To detect surface antigens, both living and paraformaldehyde-fixed cells were incubated with primary antibodies and then stained with fluorochrome-conjugated second antibodies. Antigens located within the cytoplasmic space were identified after fixation and permeabilization of cells. Live-labeled oligodendrocytes were stained brightly for myelin-oligodendrocyte glycoprotein, galactocerebroside, and other surface markers but did not stain for the PLP C-terminus or the intracellular proteins myelin basic protein and beta-tubulin. Fixation alone was sufficient for partial permeabilization of oligodendrocytes to antibodies and resulted in limited staining of the PLP C-terminus and intracellular proteins. The permeabilized oligodendrocytes stained intensely for the PLP C-terminus, myelin basic protein, and beta-tubulin. Finally, trypsinization of living oligodendrocytes eliminated surface myelin-oligodendrocyte glycoprotein staining but did not change the immunostaining properties of the PLP C-terminus. These results provide evidence that the carboxyl-terminus of PLP is located at the cytoplasmic face of oligodendroglial membranes.