Members of the myocyte enhancer factor 2 (MEF2) family of transcription factors contain highly conserved sequences within their MADS box and MEF2 domain. These motifs are required for DNA binding and dimerization properties, as well as for MEF2 association with various transcriptional activator or repressor proteins. The D-mef2 gene encodes the MEF2 protein of Drosophila and genetic studies have shown that normal D-MEF2 function is needed for muscle cell differentiation during embryogenesis and indirect flight muscle formation during pupal development. We have characterized three additional lethal alleles of D-mef2 and identified the specific mutation in each that alters a conserved amino acid present within the MADS box of all known MEF2 proteins. Mutation of these invariant residues results in the inability of mutant D-MEF2 proteins to bind DNA in vitro, muscle defects within the embryo, and adverse effects on the structure of indirect flight muscles within the adult. Since the crystal structure of a MEF2 core protein bound to DNA has been previously solved, our results correlate the mutation of specific MADS box amino acids utilized for target DNA recognition with severe myogenic phenotypes manifested during Drosophila development.