We have previously reported that high level human desmin expression depends on a 280-base pair muscle-specific enhancer which can function not only in myotubes, but can also activate gene expression in myoblasts. We report here that this enhancer contains two different regions, one active in myotubes and the other in myoblasts. In the myotube-specific region, one MyoD1 site and one MEF2 site are necessary for full enhancer activity. Site-directed mutation of the MyoD1 binding site revealed that the intact site is essential for gene expression in myotubes and for transactivation by MyoD1 or myogenin in co-transfected fibroblasts. In the myoblast-specific region, four regions are protected by nuclear factors from the myogenic cell line C2, 7; three regions contain a GC-rich sequence sharing homology with the Krox binding site. Deletion and site-directed mutation experiments demonstrated that at least two Krox-like sequences are required for enhancer activity in myoblasts. In addition, another GC-rich sequence, designated Mb, is also required for full enhancer activity in myoblasts.