In the CNS, myelin is formed from the expansion of oligodendrocyte processes. In order to study myelin assembly in the hypomyelinating mutant mouse quaking (qk), cultures of oligodendrocytes were established from affected and control animals. The cytoarchitecture of the oligodendrocytes was analyzed by performing morphometric measurements after immunostaining with antitubulin. The results indicate that the gross morphology of the processes is similar in control and mutant cells. The localization of the message for the myelin structural component, myelin basic protein (MBP), was examined by in situ hybridization. In control oligodendrocytes, 80% of MBP mRNA is found in the processes. In contrast, only 23% of MBP mRNA is localized to these structures in the mutant; the majority of MBP mRNA remains in the cell body. The mutant cells are capable of distributing mRNAs to the periphery as shown by the presence of tubulin mRNA in their processes. MBP polypeptide was visualized by immunofluorescence and found in the perikaryon, processes and membranous expansions of the control cells. In the mutant, it is largely confined to the perikaryon, reflecting the distribution of the mRNA. These results suggest that the localization of MBP polypeptide is achieved by restricting the distribution of its mRNA, and that MBP assembly into the myelin membrane occurs in the processes. This step appears to be blocked in qk oligodendrocytes in culture.