The basic helix-loop-helix (bHLH) domain defines a class of transcription factors that are essential for the regulation of many genes involved in cell differentiation and development. To determine the role of the DNA sequence in driving dimerization specificity of bHLH transcription factors, we analyzed the DNA sequence in and around a consensus hexanucleotide binding site (E-box). The bHLH domains of two transcription factors, E12 and TAL1, were chemically synthesized. The minimal DNA binding domain for both the E12 homodimer and the E12-TAL1 heterodimer was determined, thereby extending the E-box by two base pairs. Additional studies indicate that the presence of a thymine in the first flanking position 5' to the E-box prevents DNA binding of both dimer complexes. The presence of a thymine or cytosine in a flanking position two bases 5' to the E-box decreases the affinity for the E12 homodimer twofold but completely inactivates DNA binding for the E12-TAL1 heterodimer. Access to synthetic DNA and protein enabled the analysis of specific interactions between a conserved arginine residue in the basic helix of each bHLH domain and adenine in a flanking position two bases 5' to the E-box. Our results indicate a key role of the DNA sequence in driving dimerization specificity among bHLH transcription factors.