Muscle differentiation and expression of muscle-specific proteins are initiated by the binding of heterodimers of the transcription factor MyoD with E2A proteins to E-box motif d(CANNTG) in promoters or enhancers of muscle-specific genes. MyoD homodimers, however, form tighter complexes with tetraplex structures of guanine-rich regulatory sequences of some muscle genes. In this work, we identified elements in MyoD that bind E-box or tetraplex structures of promoter sequences of the muscle-specific genes alpha7 integrin and sarcomeric Mitochondrial Creatine Kinase (sMtCK). Deletions of large domains of the 315 amino acids long recombinant MyoD indicated that the binding site for both E-box and tetraplex DNA is its basic region KRKTTNADRRKAATMRERRR that encompasses the three underlined clusters of basic residues designated R(1), R(2) and R(3). Deletion of a single or pairs of R triads or R111C substitution completely abolished the E-box-binding capacity of MyoD. By contrast, the MyoD deletion mutants Delta102-114, DeltaR(3), DeltaR(1)R(3) or DeltaR(2)R(3) maintained comparable tetraplex DNA-binding capacity as reflected by the similar dissociation constants of their protein-DNA complexes. Only deletion of all three basic clusters abolished the binding of tetraplex DNA. Implications of the binding of E-box and tetraplex DNA by non-identical MyoD elements are considered.