MRF4, myogenin, MyoD, and Myf-5 are the four members of the basic helix-loop-helix family of muscle-specific regulatory factors (MRFs). We examined whether MRF4 could substitute for myogenin in vivo by determining if the myofiber- and MRF4-deficient phenotype of myogenin (-/-) mice could be rescued by a myogenin promoter-MRF4 transgene. When the transgene was expressed at a physiological level in myogenin-deficient fetuses, we found that expression of the endogenous MRF4 gene was restored to normal levels, whereas MyoD levels were unchanged. Thus, MRF4 can participate in a positive autoregulatory loop and can substitute for myogenin to activate its own promoter. Myogenin-deficient fetuses that expressed the transgene also had more myosin, more and larger myofibers, and a more normal ribcage morphology than myogenin-deficient littermates without the transgene. The transgene failed, however, to restore normal numbers of myofibers or viability to myogenin-deficient mice, because the approximately 1.6 kb myogenin promoter fragment was not expressed in most late-forming myofibers. These results demonstrate that MRF4 is able to substitute for myogenin to activate MRF4 expression and promote myofiber formation during the early stages of myogenesis.