The box 1 and 2 motif of the myelin basic protein (MBP) promoter is a potential regulatory sequence of the MBP transcription unit. A DNA fragment that contained the sequence of the box 1 and 2 motif from mouse was synthesized, and its protein binding properties were examined by gel-shift assays. The box 1 and 2 probe and nuclear extracts from mouse brain generated a pattern of six major DNA-protein complexes (a, b, c, d, e, and f). The box 1 and 2 probe and nuclear extracts from oligodendrocyte-like glioma cells 1C10 generated a pattern of DNA-protein complexes that exhibited only complexes a, b, e, and f. Complex b generated by extracts from 1C10 cells, however, was very intense compared to any of the other complexes. It was determined that dephosphorylation of the proteins in nuclear extracts from 1C10 cells with acid phosphatase significantly altered their DNA binding properties. Two proteins of minimum M, approximately 32 and approximately 38 kDa (MBP32 and MBP38) that bind to the box 1 and 2 motif were identified in these nuclear extracts by using a UV crosslinking method. MBP32 and MBP38 are found in cell types and tissues known to express the golli transcription unit of the golli-MBP gene complex and may be involved in the modulation of the MBP unit in those cells.