We have successfully established mixed glial cell primary cultures prepared from individual fetal human brains (15–18 weeks' gestation in age). Cultures were maintained for as long as 3 months in either 10% fetal calf serum (FCS) or serum-free chemically defined medium (CDM). By morphological and immunohistochemical criteria, the precursor cell for human oligodendrocytes (O-2A cell) was identified. This cell exhibited the bipolar morphology and A2B5-positive (A2B5+) immunoreactivity typical of the O-2A precursor cell. With time in culture, cells possessing a stellate morphology appeared, some of which stained with the O4 antibody, indicative of cell differentiation in the oligodendroglial lineage. At yet older culture age, arborized cells bearing the O1 (galactocerebroside, GC) immunohistochemical marker and displaying the morphological characteristics typical of more mature oligodendrocytes were found, confirming their oligodendroglial identity. Oligodendroglial differentiation was supported best by CDM rather than FCS. To complement these observations, double immunofluorescent studies were performed on parietal sections from human fetal brains at 20 to 22 weeks of gestation. Bipolar A2B5+, multipolar A2B5+/O4+, and arborized A2B5/O1+ cells were found, thus confirming the presence of oligodendrocytes in human fetal brain at this stage of prenatal development and consistent with the observations made in cell culture.