A nonradioactive in situ hybridization procedure was employed to study the distribution of mRNAs for myelin basic protein (MBP), proteolipid protein (PLP), myelin-associated glycoprotein (MAG) and 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNP) in oligodendrocytes in primary cultures of mouse brain. This procedure provided good cellular localization and allowed rapid detection of the mRNAs with low backgrounds. Gradual movement of MBP mRNAs from oligodendrocyte somas into processes was observed with time in culture. The MBP mRNAs were observed to be distributed in an asymmetric fashion within the somas and cell processes. Antigalactocerebroside staining indicated the presence of oligodendrocyte processes prior to movement of MBP mRNA, suggesting that the presence of processes alone was insufficient for translocation of MBP mRNAs. The mRNAs for PLP and MAG remained confined to the oligodendrocyte somas at all times in culture at least up to 28 days. While most of the CNP mRNAs were observed to be associated with the perikarya of oligodendrocytes, in less than 1% of these cells the presence of CNP mRNA in the processes was evident. This suggests that there may exist a subset of oligodendrocytes in which the translocation of these messages occurs.