The central nervous system expression of myelin basic protein (MBP) is restricted to oligodendrocytes and is developmentally regulated; these regulatory features are transcriptionally mediated. We have previously shown that the proximal 149 nucleotides of the MBP promoter were both necessary and sufficient to activate the transcription of MBP in cultured oligodendrocytes, but not in other cell types. Sequences within the distal portion of this promoter, which contains a nuclear factor 1 (NF1) binding site, repressed activation of the MBP promoter in Cos-7 cells, but not in oligodendrocytes. We now describe a sequence upstream of and partially overlapping the NF1 site that activates the MBP promoter in oligodendrocytes, but not in Cos-7 cells. A protein complex binds to this site, designated MEBA (myelinating glia-enriched DNA binding activity), and is enriched in nuclear extracts prepared from the brain, oligodendrocytes, and Schwann cells. The amount of MEBA parallels MBP expression and myelinogenesis in the developing brain and parallels new MBP expression as purified oligodendrocytes differentiate. Mutational analyses of binding and function distinguish MEBA, an activator, from NF1, a repressor of MBP transcription, and suggest that MEBA consists of at least two proteins. Because the binding sites of MEBA and NF1 overlap, we suggest that MEBA may either compete with or modify NF1 binding, thereby activating the MBP promoter in oligodendrocytes.