Promoter elements of the mouse myelin basic protein (MBP) gene were analyzed by in vitro transcription using HeLa cell extracts. We demonstrated the MBTE (MBP transcription element), GC-box core and TATA-box elements, at -130, -93 and -34, respectively. The TATA-box was indispensable for the promoter function. The GC-box was suggested to function co-operatively with far upstream sequences including the MBTE. The MBTE was crucial to direct maximal transcription, and also functioned with a heterologous promoter irrespective of its orientation. We identified a ubiquitous binding factor which interacted specifically with the MBTE and activated transcription. Intensive foot-printing studies demonstrated that the MBTE had a NFI-binding sequence. The MBTE was considered to be one of the strongest NFI-binding motif among known cellular genes. Interestingly, similar strong NFI-binding motifs were suggested to be present in the enhancer of JC virus whose gene is expressed like the MBP gene, in the nervous system.