The first 85 nt upstream of the transcription initiation site of the mouse desmin gene, which contain an E box (E1), the binding site of the helix-loop-helix myogenic regulators, are sufficient to confer low level muscle-specific expression. High levels of desmin expression are due to an enhancer, located between nucleotides -798 and -976, which contains an additional E box (E2) and a muscle-specific enhancer factor-2 (MEF-2) binding site. We have previously shown that both myoD and myogenin can bind to the proximal (E1) and distal (E2) boxes. Here we demonstrate that MEF-2C, a myocyte-restricted member of the MEF-2 family, can bind to the desmin MEF-2 site. Functional units for the enhancer activity required intact E2 and MEF-2 elements. The desmin enhancer can function relatively well with either the E2 box or the MEF-2 site and only mutation of both eliminates transcriptional enhancement; the presence of both of these elements is required for maximum enhancer activity. On the other hand, mutagenesis of just the proximal E1 box showed that this element is essential for desmin gene expression. Double mutations of E1 with E2 or MEF-2 sites suggested that, to achieve high levels of desmin gene expression, E1 serves most possibly as an intermediary for either E2 or MEF-2 enhancer elements to function. The location of the E1 site relative to the TATA box is crucial. Its activity is DNA turn- and distance-dependent. Furthermore, this box seems to be the main element for desmin transactivation by myoD and myogenin in 10T1/2 cells. Its inactivation diminishes the transactivation by these factors; MRF4 and Myf5, however, can still partially function, possibly by using the distal E2 box.