Cells at an intermediate stage of oligodendrocyte lineage are not only well characterized by biochemical studies but also are likely to relate to the outcome of physiological events. To elucidate the molecular events leading to the development of oligodendrocyte lineage cells, we have raised monoclonal antibodies against stage-specific immature oligodendrocytes, which have previously been isolated by a novel oligodendrocyte-lineage cell culture technique (Sakurai et al.: J Neurosci Res 52:17-26, 1998). We have isolated a mouse monoclonal antibody termed 14F7 which predominantly labels stage-specific immature oligodendrocytes and have found that the expression of 14F7 immunoreactivity in the developing neonatal rat forebrain is closely associated with cells expressing the oligodendrocyte progenitor marker A2B5 and to immature oligodendrocyte expressing O4 antigen. 14F7+ cells were distributed in the ventricular and subventricular zone and the nearby forming corpus callosum as non-myelinating cells. In contrast to cell culture observations, 14F7+ cells were seen only in oligodendrocyte lineage cells. For instance, dissociated cell culture studies indicated that 14F7 labels a cell surface molecule, and its cellular distribution is coincident with all of O4+ cells and A2B5+ cells, and even A2B5- cells. By contrast, 14F7-positive cells did not label astrocytes and, furthermore, did not label myelin basic protein (MBP)-positive oligodendrocytes. 14F7 recognized a 48-kDa protein on sodium dodecyl sulfate polyacrylamide gel electrophoresis. 14F7 immunoreactivity was detectable in rat brain as early as embryonic day 18. Furthermore, in these cells, the total time for differentiation was extended, and on maturation, these cells subsequently expressed an array of myelin-specific proteins, which normally occurs by direct contact with type-1 astrocytes. However, in the presence of 14F7, stage-specific oligodendrocytes co-cultured with astrocytes completely failed to express MBP. These data suggest that the 14F7 antigen is a novel cell surface molecule that is expressed in the intermediate stage of oligodendrocyte-lineage cells, and it is expected that it regulates the differentiation of oligodendrocyte throughout development.