Previous investigations have shown that expression of the muscle-specific intermediate filament desmin gene in skeletal muscle is controlled in part by a 5' muscle-specific enhancer. This enhancer activity can be divided into myoblast-specific and myotube-specific activation domains. The myotube-specific region contains a MyoD and MEF2 sites, whereas the myoblast-specific region contains Sp1, Krox, and Mb sites. In the present study, we designed mutations in the conserved portion of the myotube-specific region; transfection analysis of these mutations showed that a novel site located between the MyoD and MEF2 sites, named Mt (GGTATTT), is required for full transcriptional activity of the desmin enhancer in skeletal muscle. Although gel mobility shift assays demonstrate that myotube, myoblast, fibroblast, and HeLa nuclear extracts contain a nuclear factor that binds specifically to Mt, four copies of the Mt site function as the native enhancer only in myotubes. Functional synergism among the MyoD, MEF2, and Mt sites in myotubes has been demonstrated. These results show that the novel Mt site cooperates with MyoD and MEF2 to give maximal expression of the desmin gene.