Phenotypic expression and proliferative capacity of the cells of oligodendrocyte lineage were investigated in primary cultures isolated from fetal human brains of 12-15 weeks' gestation using double immunolabeling with Ranscht-monoclonal antibody (R-mAb) or O4 and antibromodeoxyuridine (BrdU) antibody. Cultured cells of oligodendrocyte lineage consisted of a major population of R-mAb+O4- cells and minor populations of R-mAb-O4+ and R-mAb+O4+ cells. Most of the R-mAb+O4- cells exhibited a uni-, bi-, or tripolar immature morphology, while the majority of the R-mAb+O4+ cells exhibited a multipolar mature morphology. R-mAb-O4+ cells contained a mixture of immature and mature cell types. When incubated in serum-free culture medium containing BrdU for 4 days, 42% of total oligodendrocytes expressed nuclear BrdU immunolabeling. R-mAb+ cells exhibited a higher degree of BrdU immunolabeling, indicating that they have greater capacities for proliferation than O4+ cells. The large majority of BrdU+ cells exhibited an immature morphology. Inclusion of insulin, insulin-like growth factor (IGF)-I, basic fibroblast growth factor (bFGF), or fetal bovine serum in culture medium did not stimulate proliferation of oligodendrocytes, while platelet-derived growth factor (PDGF) or PDGF plus bFGF increased the number of R-mAb+BrdU+ and O4+BrdU+ cells over control, even though the results were not statistically significant. In addition, insulin and IGF-I induced a 3-fold increase in the number of R-mAb+O4+ cells, indicating that they promoted differentiation of oligodendrocytes. The present study indicates that fetal human oligodendrocytes in culture exhibit a considerable degree of proliferative capacity without requirement of exogenous growth factors and that both insulin and IGF-I promote their differentiation.