The myelin basic protein (MBP) gene is expressed only in oligodendrocytes and Schwann cells, and expression follows a tightly regulated developmental time course. Cell type- and developmental stage-specific expression of the MBP gene appears to be regulated by a series of cis-acting elements located upstream of the transcription start site. The proximal element of the MBP regulatory region (MB1), located between nucleotides -14 and -50, is one of several elements participating in the programmed expression of MBP. In this report, we describe the molecular cloning and characterization of myelin gene expression factor-2 (Myef-2), a protein isolated from mouse brain that binds specifically to single-stranded DNA derived from the MB1 element and represses transcription of the MBP gene in transient transfection assay. Myef-2 mRNA is developmentally regulated in mouse brain; its peak expression occurs at postnatal day 7, prior to the onset of MBP expression. The developmental pattern of Myef-2 mRNA expression coincides with that previously described for SCIP, a POU domain transcription factor that also represses myelin basic protein expression. The myef-2 gene maps to mouse chromosome 2. The relevance of these findings for regulation of MBP gene expression and oligodendrocyte differentiation is discussed.