The iron transport glycoprotein, transferrin (Tf), localizes exclusively in oligodendrocytes in brain tissue sections. Previously, we showed that Tf is also expressed in oligodendrocytes in primary cultures established from newborn rat brains. Its developmental appearance precedes that of galactocerebroside (GC). In this study, Tf expression in primary brain cell cultures was investigated over a 4-week period in relation to GC and myelin basic protein (MBP), respectively, early and late markers of oligodendrocyte development. From 9 days in vitro and thereafter, all Tf+ cells were also found to be GC+. With increasing age the number of Tf+ cells decreased while the number of MBP+ cells increased. However, less than 10% of oligodendrocytes co-expressed Tf and MBP at any age. MBP+ cells were largely found in cell clusters which increased in size and number with age in culture. Interestingly, Tf+ cells were located around the clusters of MBP+ cells which displayed elaborate branched processes. The transient expression of Tf in oligodendrocytes which become MBP+, suggests a role for Tf in the early stages of myelinogenesis. The results also demonstrate the existence of three phenotypically distinct populations of oligodendrocytes. A new model of developmental and functional subpopulations of oligodendrocytes is proposed.