Myogenic transcription is activated by the binding of heterodimers of the basic helix-loop-helix proteins MyoD and E12 or E47 to a consensus E-box sequence, d(CANNTG), in promoter or enhancer regions of muscle-specific genes. Homodimers of MyoD bind E-box less tightly and are less efficient activators of transcription. Recent results from our laboratory (Yafe, A., Etzioni, S., Weisman-Shomer, P., and Fry, M. (2005) Nucleic Acids Res. 33, 2887-2900) indicate that regulatory sequences of several muscle-specific genes contain a disproportionate high content of guanine clusters that readily form hairpin and parallel-stranded unimolecular and bimolecular tetraplex structures. Here we have shown that homodimers of full-length recombinant MyoD formed complexes with bimolecular tetraplex structures of muscle-specific regulatory sequences but not with their double-stranded, hairpin, or unimolecular tetraplex forms. Preferential binding of homodimeric MyoD to bimolecular tetraplex DNA structures over E-box DNA was reflected by the 18.7-39.9-fold lower dissociation constants, Kd, of the MyoD-tetraplex DNA complexes. Conversely, MyoD-E47 heterodimers formed tighter complexes with E-box as indicated by their 6.8-19.0-fold lower Kd relative to complexes with bimolecular tetraplex DNA structures. Similarly, homodimers of the 60-amino acid basic helix-loop-helix domain of MyoD bound E-box more efficiently and tetraplex DNA less efficiently than homodimers of full-length MyoD. It might be that the favored binding of MyoD homodimers to tetraplex DNA structures lowers their ability to activate muscle-specific gene transcription, whereas the formation of MyoD-E47 heterodimers and their preferential binding to E-box DNA enhance transcription.