The serum response factor (SRF) and myocyte enhancer factor 2A (MEF2A) represent two human members of the MADS-box transcription factor family. Each protein has a distinct biological function which is reflected by the distinct specificities of the proteins for coregulatory protein partners and DNA-binding sites. In this study, we have investigated the mechanism of DNA binding utilized by these two related transcription factors. Although SRF and MEF2A belong to the same family and contain related DNA-binding domains, their DNA-binding mechanisms differ in several key aspects. In contrast to the dramatic DNA bending induced by SRF, MEF2A induces minimal DNA distortion. A combination of loss- and gain-of-function mutagenesis identified a single amino acid residue located at the N terminus of the recognition helices as the critical mediator of this differential DNA bending. This residue is also involved in determining DNA-binding specificity, thus indicating a link between DNA bending and DNA-binding specificity determination. Furthermore, different basic residues within the putative recognition alpha-helices are critical for DNA binding, and the role of the C-terminal extensions to the MADS box in dimerization between SRF and MEF2A also differs. These important differences in the molecular interactions of SRF and MEF2A are likely to contribute to their differing roles in the regulation of specific gene transcription.