The myogenic determination gene MyoD contains a 60 amino acid domain that is necessary for both sequence-specific DNA binding and myogenic conversion of transfected C3H10T1/2 mouse embryo fibroblasts. We have generated deletion, insertion, and substitution mutants to probe the structure and function of this region, both in vitro and in vivo. Our results are consistent with a previous proposal that a helix-loop-helix (HLH) motif mediates protein dimerization. A highly basic region located immediately upstream of the HLH motif is required for DNA binding, which occurs if and only if the HLH motif is capable of dimerization. All mutants of MyoD that do not bind DNA in vitro fail to activate myogenesis or expression of a co-transfected muscle-specific reporter gene in transfected C3H10T1/2 cells. Replacing either helix 1, helix 2, or the loop sequence of MyoD with the analogous sequence of the Drosophila T4 achaete-scute protein (which is required for neurogenesis) has no substantial effect on DNA binding or biological activity. However, replacing the basic region of MyoD with the analogous sequence of the kappa immunoglobulin enhancer binding protein E12, or the T4 achaete-scute protein, produces proteins still capable of specific DNA binding in vitro, yet without significant biological activity. These findings suggest that within the 13 amino acid sequence of the MyoD basic region lies a recognition code that determines muscle-specific gene expression, although specific DNA binding per se is not sufficient to activate the muscle program.