The box I and II motif located within the promoter of the myelin basic protein gene contains a simian virus 40 T-antigen-binding site, a MyoD/E2a-binding site, and a glucocorticoid receptor-binding site. We have found proteins within nuclear extracts from adult mouse brain, rat embryonic cerebral cortex in culture, and a mouse oligodendrocyte-like cell line that bind to a 32P-labeled synthetic DNA fragment containing the sequences of the box I and II motif. Three major complexes (A, B, and C) were seen in gel-shift assays. Only complexes A and B were competed out by the unlabeled box I and II fragment or by another synthetic DNA fragment that also contains sequences similar to a glucocorticoid receptor-binding site. Therefore, complexes A and B were thought to be specific. The expression pattern of the proteins responsible for the formation of these complexes was also assessed during development in mouse brain.