We measured transiently-expressed beta-galactosidase activity by introducing the mouse myelin basic protein (MBP)-lacZ chimeric gene (MBP-lacZ) into the NG108-15 neuronal/glial hybrid cell line. Deletion studies of the promoter region of the MBP gene showed that the promoter region between -1318 bp and -254 bp might contain sequences that repress MBP promoter activity. Fine deletion analysis using BAL 31 exonuclease revealed sequences between bp -208 and -140, -139 and -118, and -89 and -75 which were critical for promoter activity in NG 108-15 cells. DNaseI footprinting analysis revealed a cellular factor(s) that bind to the promoter region between bp -127 and -106 with NG108-15 whole cell extracts. The SV40 promoter was activated by insertion of the sequences around the region protected in footprinting experiments, in a manner independent of its orientation in NG108-15 cells. This protected region is thought to be one of the critical cis-acting DNA elements for efficient transcription.