Using differential adhesion we successfully isolated relatively pure populations of mouse oligodendrocytes which can be maintained in vitro for more than two months. The highest percentage of galactocerebroside (GalC)-positive oligodendrocytes was 95% at 3 days after isolation. Thereafter, proliferation of astrocytes and fibroblasts occurred more quickly than did oligodendrocyte precursor division. GalC-positive oligodendrocytes rarely incorporate [3H]thymidine so that the use of a mitotic inhibitor (5 X 10(-6)M AraC) reduced the number of non-oligodendrocytes so as to maintain the purity of oligodendrocytes at more than 75% for 14 days in culture. This system will be of use for immunological and virological studies which require viable cultured mouse oligodendrocytes.