myogenin (-/-) mice display severe skeletal muscle defects despite expressing normal levels of MyoD. The failure of MyoD to compensate for myogenin could be explained by distinctions in protein function or by differences in patterns of gene expression. To distinguish between these two possibilities, we compared the abilities of constitutively expressed myogenin and MyoD to support muscle differentiation in embryoid bodies made from myogenin (-/-) ES cells. Differentiated embryoid bodies from wild-type embryonic stem (ES) cells made extensive skeletal muscle, but embryoid bodies from myogenin (-/-) ES cells had greatly attenuated muscle-forming capacity. The inability of myogenin (-/-) ES cells to generate muscle was independent of endogenous MyoD expression. Skeletal muscle was restored in myogenin (-/-) ES cells by constitutive expression of myogenin. In contrast, constitutive expression of MyoD resulted in only marginal enhancement of skeletal muscle, although myocyte numbers greatly increased. The results indicated that constitutive expression of MyoD led to enhanced myogenic commitment of myogenin (-/-) cells but also indicated that committed cells were impaired in their ability to form muscle sheets without myogenin. Thus, despite their relatedness, myogenin's role in muscle formation is distinct from that of MyoD, and the distinction cannot be explained merely by differences in their expression properties.