MyoD is a skeletal muscle-specific protein that is able to induce myogenesis in a wide variety of cell types. In this report, we show that MyoD is a DNA binding protein capable of specific interaction with two regions of the mouse muscle creatine kinase gene upstream enhancer, both of which are required for full muscle-specific enhancer activity. MyoD shares antigenicity and DNA binding specificity with MEF1, a myocyte-specific DNA binding factor. The contiguous basic and myc homology regions of MyoD that are necessary and sufficient for specific DNA interaction are the same regions of the protein required to convert 10T1/2 fibroblasts into muscle. These findings suggest that the biological activity of MyoD is mediated via its capacity for specific DNA interaction.