The changes in the ultrastructure of oligodendrocytes with time in culture were examined by transmission electron microscopy. Oligodendrocytes were isolated from ovine white matter and maintained in Dulbecco's modified Eagle's medium supplemented with 20% horse serum. One day after being placed in plastic culture dishes, most cells aggregate into clusters that do not attach to the surface for several days. Cells within these clusters form junctional complexes resembling gap junctions, their nuclear chromatin becomes uniformly dispersed, they are rich in polyribosomes, and their cytoplasm is less osmiophilic than oligodendrocytes in ovine white matter or cells fixed immediately after isolation. After several days in culture, the cells become attached to the plastic surface, chromatin once again becomes clumped beneath the nuclear membrane, rough endoplasmic reticulum accumulates within the cytoplasm and the cells extend long processes containing cytoplasmic organelles. From these observations, it appears that oligodendrocytes undergo a transient dedifferentiation during their recovery from the trauma of isolation, not unlike the central chromatolysis seen in neurons following axonal injury. After acclimatization to tissue culture conditions, isolated oligodendrocytes show many of the morphologic features seen in vivo and therefore offer a good model to study myelination.