The plp gene encodes the proteolipid protein and its alternatively spliced product DM-20, major proteins of CNS myelin. In the mouse, plp/dm-20 transcripts are expressed beginning at embryonic day 9.5 (E9.5) by restricted foci of germinative neuroepithelial cells. To determine the identity of the neural precursors expressing plp/dm- 20, a zeomycin resistance gene fused to the lacZ reporter was expressed in transgenic mice under the control of the plp regulatory sequences. In the three different lines generated, the pattern of β-galactosidase expression was similar and superimposable on the expression pattern of endogenous plp/dm-20. Bothin vivo and in vitro, the transgene was expressed by O4⁺ pre-oligodendrocytes, and later by RIP⁺ differentiated oligodendrocytes, but not by neuronal cells, astrocytes, or radial glial cells. After zeomycin selection, a dramatic enrichment in O4⁺pre-oligodendrocytes was observed in cultures derived from E12.5 transgenic embryos. This enrichment indicates the oligodendroglial specification of neural precursors that continuously expressplp/dm-20. Early plp/dm-20-expressing precursors, however, appear to be a separate population from previously described PDGFRα oligodendrocyte precursors, as shown by the striking differences in their (1) patterns of distribution and (2) responsiveness to PDGF. These data suggest that oligodendrocytes have a plural origin and that early plp/dm-20 defines one of the neural lineages generating oligodendrocytes.