The monoclonal antibody O4 has been used to define a biologically distinct stage of the oligodendroglial lineage in vitro. Furthermore, O4+ oligodendroglial progenitors have been found in cell cultures derived from mature tissue, leading to speculation about the presence of oligodendroglial progenitors in the adult central nervous system (CNS). However, the existence of adult oligodendroglial progenitors has yet to be conclusively demonstrated in the intact animal. We have investigated the expression of O4 immunoreactivity in the developing and mature rat forebrain and the relationship of these cells to cells expressing the early oligodendroglial progenitor markers GD3 ganglioside and NG2 chondroitin sulfate proteoglycan, and to differentiated galactocerebroside expressing oligodendroglia. By the day of birth O4+ cells were already widely distributed throughout the formative corpus callosum and increased in number in the white matter and cortical gray matter over the first 2 postnatal weeks. In contrast to cell culture observations, most O4+ cells seen over this period failed to express GD3, although the majority did express NG2. Beginning at postnatal day 4, NG2+/O4-progenitors in the corpus callosum and cerebral cortical gray matter underwent a wave of differentiation into NG2+/O4+ cells and then into galactocerebroside-positive oligodendroglia. Interestingly, not all cells underwent this progression: a population remained as O4+/NG2+ progenitors. Furthermore, this O4+/NG2+ population persisted into adulthood and failed to express their GD3, galactocerebroside, RIP, or myelin basic protein (MBP). They were also distinguishable from glial fibrillary acidic protein+ and glutamine synthetase+ astrocytes and OX-42+ microglia. We therefore propose that these O4+/NG2+ cells represent adult oligodendroglial progenitors hitherto only described in cell culture.