We have prepared highly purified cultures of rat oligodendrocytes by a modification of the procedure of McCarthy and de Vellis [1980]. By utilizing a substratum derived from lysed glia and a calf serum-containing medium with a high concentration of transferrin, the oligodendrocyte cultures display a high degree of purity, the ability to survive several months of culture, and a striking ability to produce a myelin-like membrane. We have examined the production of this myelin-like membrane using immunocytochemical and biochemical probes as well as an extensive morphological examination at the electron microscopic level. The membrane appears to be produced in a similar developmental pattern to that observed in vivo and it has the structural characteristics of loosely packed central nervous system myelin.